WO2025237408A1 - Modified nucleoside, double-stranded rna molecule comprising same, and use - Google Patents

Abstract

The present invention relates to a modified nucleoside, and a double-stranded RNA molecule comprising the modified nucleoside in a seed region of an antisense strand. At least one modified nucleoside is introduced into the seed region of the antisense strand of the double-stranded RNA molecule, such that a target gene is silenced, and the off-target effect of the double-stranded RNA molecule is suppressed. The present invention further relates to a use of the modified nucleoside in preparation of a double-stranded RNA molecule, and a pharmaceutical composition containing the double-stranded RNA molecule, used for preventing and treating a disease.

Description

Modified nucleosides, double-stranded RNA molecules containing them, and their uses Technical Field

This invention provides a class of modified nucleosides, their analogues, and oligomers prepared from them. More specifically, this invention provides modified nucleosides and their analogues that can be incorporated into oligomers, particularly dsRNA molecules. Placing at least one of these modified nucleosides and their analogues in the seed region of the antisense strand of a dsRNA molecule can reduce the thermal stability of the antisense strand seed region, thereby reducing off-target effects on non-target genes.

Background Technology

Oligonucleotides are polymers of nucleotides (RNA, DNA, and their analogues). Nucleic acid inhibitor molecules are oligonucleotides that regulate intracellular RNA levels and have shown early promise in the treatment of genetic diseases, metabolic diseases, cancer, and viral infections. Nucleic acid inhibitor molecules can regulate RNA expression through a different set of mechanisms, including RNA interference (RNAi).

RNAi is a conserved pathway found in most eukaryotes, in which double-stranded RNA (dsRNA) molecules can suppress the expression of target genes complementary to the dsRNA. In a typical RNAi pathway, the longer dsRNA molecule is cleaved by the enzyme Dicer into shorter RNA duplexes (also known as “small interfering RNA,” siRNA). It has been shown that siRNA binds to Dicer, trans-activating response RNA-binding protein (TRBP), and Argonaute 2 (Ago2) to form a complex known as the RNA-induced silencing complex (RISC). Ago2 is a nuclease that uses the antisense strand (also known as the guide strand) of the siRNA to guide the sequence-specific cleavage of the target mRNA.

Various siRNA molecules have been developed over the years. For example, early work on RNAi inhibitor molecules focused on double-stranded nucleic acid molecules mimicking native siRNAs, where each strand has 19-25 nucleotides and includes at least one 3' overhang with 1 to 5 nucleotides (see, for example, U.S. Patent No. 8,372,968). Subsequently, longer dsRNA molecules were developed, which are cleaved in vivo by cleavage enzymes into active RNAi inhibitor molecules (see, for example, U.S. Patent No. 8,883,996). Subsequent work developed extended double-stranded nucleic acid inhibitor molecules, where at least one end of at least one strand extends beyond the double-stranded target region of the molecule, including one of the strands having a thermodynamically stable tetracyclic structure (see, for example, U.S. Patent Nos. 8,513,207, 8,927,705, WO2010/033225, and WO2016100401). These structures include single-stranded extensions (on one or both sides of the molecule) and double-stranded extensions.

In recent years, modified nucleosides have been increasingly used in siRNA, for example, modifications to the sugar ring of ribonucleic acid, such as 2'-fluoro (2'-F) modification, 2'-O-methyl (2'-OMe) modification, 2'-O-methoxyethyl (2'-O-MOE) modification, non-locked nucleotide (UNA) modification, locked nucleotide (LNA) modification, 4'-CH( _CH3 )-O-2'(cEt) modification, and diol nucleic acid (GNA) modification; and the replacement of 5'-phosphate esters with phosphatase resistance analogs, such as 5'-(E)-vinylphosphonate (5'-(E)-VP). In some cases, chemical modifications have been introduced into nucleic acid inhibitor molecules to introduce properties that may be desired under specific conditions, such as those experienced after in vivo administration. Introducing specific modified nucleosides at specific positions in the sequence has been shown to increase the double-strand stability of siRNA, or increase its resistance to nuclease degradation, or increase its cellular uptake, or increase its inhibitory activity against target genes, or increase its specificity against target mRNA.

Off-target effects of siRNA are a major cause of the toxic side effects of siRNA drugs. The most significant off-target effect is the microRNA (miRNA)-like effect, where the core Ago protein in RNA interference treats siRNA as miRNA (Lam et al., Molecular Therapy Nucleic Acids (2015) 4, e252). miRNAs primarily recognize target genes through base pairing between their seed region (positions 2-8 from the 5' end) and target mRNA. Therefore, off-target effects caused by miRNAs originate from the complementarity of the seed region of the RISC antisense strand with bases of one or more target mRNAs. MiRNA-like off-target effects in siRNA have been reported in several studies. The sequence of the seed region in siRNA affects the expression of multiple genes, and is severe enough to cause up to 30% positive responses in siRNA-based phenotypic screening. Furthermore, in the case of miRNAs, when the interaction between the seed region and the target weakens, they have also been reported to silence the target mRNA through compensatory pairing (3'-compensatory pairing) in their 3' terminal regions, suggesting that miRNA-like off-target effects may be regulated by this mechanism (Janas et al., Nat. Commun. (2018) 9, 723).

Therefore, the purpose of this invention is to eliminate or reduce the miRNA-like off-target effect of siRNA by applying modified nucleosides without affecting the gene silencing effect of siRNA on target mRNA genes.

Summary of the Invention

This invention develops a novel nucleoside compound with a novel structure. When applied to dsRNA sequences, especially when placed in the seed region of the antisense strand (i.e., positions 2-8 at the 5' end of the antisense strand, counting from the 5' end), the corresponding dsRNA can effectively reduce the off-target effects of oligonucleotide sequences while retaining the inhibitory activity on the target mRNA.

This invention provides effective nucleotide or chemical motifs of dsRNA that can inhibit target gene expression while having reduced off-target gene silencing effects, and provides RNAi compositions suitable for therapeutic use.

In a first aspect, the present invention provides a compound of formula (I).

in:

R ₁ is selected from halogen, alkyl, -O-alkyl, -S-alkyl and -NH-alkyl, wherein the alkyl is optionally substituted by one or more substituents selected from the following: hydroxyl, cyano, halogen, -O-alkyl, -S-alkyl, -NH ₂ , -NH-alkyl and -N(alkyl) ₂ ;

_R2 is selected from H, halogens, alkyl groups, and -O-alkyl groups;

R ₃ is selected from -O-DMTr or -CH ₂ -X-DMTr, where X is O, S or NH, and DMTr represents 4,4'-dimethoxytriphenylmethyl;

Where Base is a natural or non-natural base, or a protected natural or non-natural base.

In one embodiment, the compounds of the present invention are selected from compounds of formulas (Ia) and (Ib):

_R1 , _R2 , _R3 and Base are defined in the compounds of formula (I).

In one embodiment, the present invention provides compounds of formula (I), formula (Ia) and formula (Ib), wherein the alkyl group is selected from _C1 - _C30 alkyl groups, such as _C1 - _C10 alkyl groups, _C1 - _C6 alkyl groups, _C1 - _C4 alkyl groups, and the alkyl group is straight-chain or branched.

In one embodiment, the present invention provides compounds of formula (I), formula (Ia) and formula (Ib), wherein: _R1 is selected from halogen, _C1 - _C6 alkyl, -O-( _C1 - _C6 alkyl), -S-( _C1 - _C6 alkyl), and -NH-( _C1 - _C6 alkyl), wherein the _C1 - _C6 alkyl is optionally substituted by one or more substituents selected from: hydroxyl, cyano, halogen, -O-( _C1 - _C6 alkyl), -S-( _C1 - _C6 alkyl), _-NH2 , -NH-( _C1 - _C6 alkyl), and -N( _C1 - _C6 alkyl) ₂ ;

_R2 is selected from H, halogens, _C1 - _C6 alkyl groups and -O-( _C1 - _C6 alkyl groups);

R ₃ is selected from -O-DMTr or -CH ₂ -X-DMTr, where X is O, S or NH, and DMTr represents 4,4'-dimethoxytriphenylmethyl;

Where Base is a natural or non-natural base, or a protected natural or non-natural base.

In one embodiment, the present invention provides compounds of formulas (I), (Ia), and (Ib), wherein: _R1 is selected from halogens, _C1 - _C6 alkyl groups, and -O-( _C1 - _C6 alkyl groups), wherein the _C1 - _C6 alkyl group is optionally substituted with a substituent selected from: hydroxyl, halogen, -O-( _C1 - _C6 alkyl), -S-( _C1 - _C6 alkyl), _-NH2 , -NH-( _C1 - _C6 alkyl), and -N( _C1 - _C6 alkyl) ₂ ; preferably, _R1 is selected from F, methoxy, and methyl groups optionally substituted with hydroxyl, halogen, -O-( _C1 - _C6 alkyl), -S-( _C1 - _C6 alkyl), and -NH-( _C1 - _C6 alkyl); more preferably, _R1 is selected from _F , _-CH3 , _-OCH3 , -CH2-OH, _-CH2F , and _-CH2. -OCH ₃ , -CH ₂ -SCH ₃ , -CH ₂ -NH(CH ₃ ).

In one embodiment, the present invention provides compounds of formula (I), (Ia) and (Ib), wherein: _R2 is selected from H, _C1 - _C6 alkyl and -O-( _C1 - _C6 alkyl); preferably, _R2 is selected from H, methyl and methoxy; more preferably, _R2 is H.

In one embodiment, the present invention provides compounds of formula (I), formula (Ia) and formula (Ib), wherein _R1 and _R2 are not H.

In one embodiment, the present invention provides compounds of formula (I), formula (Ia) and formula (Ib), wherein _R1 is selected from halogens, _C1 - _C6 alkyl and -O-( _C1 - _C6 alkyl), and _R2 is selected from _C1 - _C6 alkyl and -O-( _C1 - _C6 alkyl), wherein the _C1 - _C6 alkyl is optionally substituted by a substituent selected from: hydroxyl, halogen, -O-( _C1 - _C6 alkyl), -S-( _C1 - _C6 alkyl), _-NH2 , -NH-( _C1 - _C6 alkyl) and -N(C1- _C6 alkyl) _{2 .}

In one embodiment, the present invention provides compounds of formula (I), formula (Ia) and formula (Ib), wherein _R1 is selected from F, methoxy, and methyl groups optionally substituted with hydroxyl, halogen, -O-( _C1 - _C6 alkyl), -S-( _C1 - _C6 alkyl), and -NH-( _C1 - _C6 alkyl), and _R2 is selected from _C1 - _C6 alkyl and -O-( _C1 - _C6 alkyl).

In one embodiment, the present invention provides compounds of formulas (I), (Ia), and (Ib), wherein _R1 is selected from F, _-CH3 , _-OCH3 , _-CH2 -OH, _-CH2F , -CH2- _OCH3 , _{-CH2 - SCH3} , and _-CH2 -NH( _CH3 ), and _R2 is selected from methyl and methoxy. Preferably, _R1 is selected from F, _-CH3 , and _-OCH3 , and _R2 is selected from methyl.

In one embodiment, the present invention provides compounds of formula (I), formula (Ia) and formula (Ib), wherein: _R3 is selected from -O-DMTr or _-CH2 -O-DMTr.

In one embodiment, the present invention provides compounds of formula (I), formula (Ia) and formula (Ib), wherein: Base is selected from natural bases A, U, C, G, T or protected natural bases A, U, C, G, T.

In one embodiment, the present invention provides compounds of formula (I), formula (Ia) and formula (Ib), wherein: Base is selected from non-natural purine bases.

In one embodiment, the present invention provides compounds of formula (I), formula (Ia) and formula (Ib), wherein: Base is selected from non-natural pyrimidine bases.

In one embodiment, the non-natural purine base is independently selected from the following bases:

In one embodiment, the non-natural pyrimidine base is independently selected from the following bases:

In one embodiment, the present invention provides the following compound,

R is methyl or C2-C30 alkyl, Base is as defined above, preferably selected from A, U, G, C, T or protected A, U, C, G, T, and stereochemically is R or S, and for unspecified chiral centers it is a combination of R and S.

In one embodiment, the present invention provides the following compound,

The Base is selected from A, U, G, C, T or protected A, U, C, G, T.

In one embodiment, the present invention provides the following compound,

Wherein Base is as defined above, preferably Base is selected from A, U, G, C, T or protected A, U, C, G, T.

In another aspect, the present invention provides a compound of formula (II).

in:

R ₁ is selected from halogen, alkyl, -O-alkyl, -S-alkyl and -NH-alkyl, wherein the alkyl is optionally substituted by one or more substituents selected from the following: hydroxyl, cyano, halogen, -O-alkyl, -S-alkyl, -NH ₂ , -NH-alkyl and -N(alkyl) ₂ ;

R ₃ is selected from -O-DMTr or -CH ₂ -X-DMTr, where X is O, S or NH, and DMTr represents 4,4'-dimethoxytriphenylmethyl;

R ₄ is selected from halogens, alkyl groups, and -O-alkyl groups;

Where Base is a natural or non-natural base, or a protected natural or non-natural base.

In one embodiment, the compound of formula (II) of the present invention is selected from compounds of formulas (IIa) and (IIb).

In one embodiment, the present invention provides compounds of formula (II), formula (IIa) and formula (IIb), wherein the alkyl group is selected from _C1 - _C30 alkyl groups, such as _C1 - _C10 alkyl groups, _C1 - _C6 alkyl groups, _C1 - _C4 alkyl groups, and the alkyl group is straight-chain or branched.

In one embodiment, the present invention provides compounds of formula (II), formula (IIa) and formula (IIb), wherein: _R1 is selected from halogen, _C1 - _C6 alkyl, -O-( _C1 - _C6 alkyl), -S-( _C1 - _C6 alkyl), and -NH-( _C1 - _C6 alkyl), wherein the _C1 - _C6 alkyl is optionally substituted by one or more substituents selected from: hydroxyl, cyano, halogen, -O-( _C1 - _C6 alkyl), -S-( _C1 - _C6 alkyl), _-NH2 , -NH-( _C1 - _C6 alkyl), and -N( _C1 - _C6 alkyl) ₂ ;

R ₃ is selected from -O-DMTr or -CH ₂ -X-DMTr, where X is O, S or NH, and DMTr represents 4,4'-dimethoxytriphenylmethyl;

_R4 is selected from halogens, _C1 - _C6 alkyl groups, and -O-( _C1 - _C6 alkyl groups);

Where Base is a natural or non-natural base, or a protected natural or non-natural base.

In one embodiment, the present invention provides compounds of formula (II), formula (IIa) and formula (IIb), wherein: _R1 is selected from halogen, _C1 - _C6 alkyl and -O-( _C1 - _C6 alkyl), said _C1 - _C6 alkyl optionally substituted with a substituent selected from: hydroxyl, halogen, -O-( _C1 - _C6 alkyl), -S-( _C1 - _C6 alkyl), _-NH2 , -NH-( _C1 - _C6 alkyl) and -N( _C1 - _C6 alkyl) ₂ ; preferably, _R1 is selected from F, methoxy and methyl optionally substituted with hydroxyl, halogen, -O-( _C1 - _C6 alkyl), -S-( _C1 - _C6 alkyl) and -NH-( _C1 - _C6 alkyl); more preferably, _R1 is selected from _-CH3 .

In one embodiment, the present invention provides compounds of formula (II), formula (IIa) and formula (IIb), wherein: _R4 is selected from _C1 - _C6 alkyl and -O-( _C1 - _C6 alkyl); preferably, _R4 is selected from methyl.

In one embodiment, the present invention provides compounds of formula (II), formula (IIa) and formula (IIb), wherein: _R3 is selected from -O-DMTr or _-CH2 -O-DMTr.

In one embodiment, the present invention provides compounds of formula (II), formula (IIa) and formula (IIb), wherein: Base is selected from natural bases A, U, C, G, T or protected natural bases A, U, C, G, T.

In one embodiment, the present invention provides compounds of formula (II), formula (IIa) and formula (IIb), wherein: Base is selected from non-natural purine bases.

In one embodiment, the present invention provides compounds of formula (II), formula (IIa) and formula (IIb), wherein: Base is selected from non-natural pyrimidine bases.

In one embodiment, the present invention provides the following compound,

Wherein Base is as defined above, preferably Base is selected from A, U, G, C, T or protected A, U, C, G, T.

In one specific embodiment, the present invention provides the following compound,

In some embodiments, the modified nucleoside is placed at positions 5 to 8 of the antisense (AS) chain, preferably at positions 6 to 8 of the antisense (AS) chain.

In double-stranded RNA, the corresponding sense (SS) strand position has a specially modified nucleoside that pairs with it, such as DNA, 2’-OMe, 2’-F, 2’-O-MOE, threonine (TNA), etc.

In a second aspect, the present invention provides a double-stranded RNA molecule comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand and the antisense strand each have 14-40 nucleotides, for example, 15 to 30, for example, 17 to 25, and 19 to 23 nucleotides, respectively, wherein the antisense strand is sufficiently complementary to a target sequence to mediate RNA interference, wherein the antisense strand comprises at least one compound of the first aspect of the present invention at nucleotide positions 2-8 (e.g., positions 5-8; more preferably positions 6-8) of the 5' region (counting from the 5' end).

In some embodiments, the present invention provides a double-stranded RNA molecule comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand and the antisense strand each have 14-40 nucleotides, for example, 15-30, for example, 17-25, and 19-23 nucleotides, respectively, wherein the antisense strand is sufficiently complementary to a target sequence to mediate RNA interference, wherein the nucleotide at positions 2-8 (e.g., positions 5-8; more preferably, positions 6-8) of the 5' region of the antisense strand has one of the following structures:

in,

R ₁ is selected from halogen, alkyl, -O-alkyl, -S-alkyl and -NH-alkyl, wherein the alkyl is optionally substituted by one or more substituents selected from the following: hydroxyl, cyano, halogen, -O-alkyl, -S-alkyl, -NH ₂ , -NH-alkyl and -N(alkyl) ₂ ;

_R2 is selected from H, halogens, alkyl groups, and -O-alkyl groups;

R ₄ is selected from halogens, alkyl groups, and -O-alkyl groups;

Base is a natural or non-natural base, or a protected natural or non-natural base, as defined above.

In some embodiments, the present invention provides a double-stranded RNA molecule comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand and the antisense strand each have 14-40 nucleotides, for example, 15-30, for example, 17-25, and 19-23 nucleotides, respectively, wherein the antisense strand is sufficiently complementary to a target sequence to mediate RNA interference, wherein the nucleotide at positions 2-8 (e.g., positions 5-8; more preferably, positions 6-8) of the 5' region of the antisense strand has one of the following structures:

_R1 , _R2 , _R4 , and Base are defined as described above.

In some embodiments, the sense strand of the double-stranded RNA molecule of the present invention further includes a ligand attached to either end of the sense strand, the ligand being covalently linked to the sense strand via a linker. For example, the ligand is an asialic acid glycoprotein receptor (ASGPR) ligand, and the ASGPR ligand comprises one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent linker.

In some embodiments, the sense strand of the double-stranded RNA molecule of the present invention has a modified nucleoside, such as DNA, 2’-OMe, 2’-F, 2’-O-MOE, or TNA, at a position that pairs with the nucleotide position of the compound of the first aspect of the present invention on the antisense strand.

In some embodiments, substantially all nucleotides of the antisense strand and/or sense strand of the double-stranded RNA molecule of the present invention are modified nucleotides. Preferably, the sense strand and/or antisense strand comprises at least one modified nucleotide selected from: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-O-methoxyethyl modified nucleotides, and deoxyribonucleotides. In some specific embodiments, the double-stranded RNA molecule of the present invention comprises paired sense and antisense strands as shown in Table 2B or Table 5.

In a third aspect, the present invention provides a pharmaceutical composition comprising the double-stranded RNA molecule described in the second aspect of the present invention and a pharmaceutically acceptable excipient.

In a fourth aspect, the present invention provides a gene silencing kit comprising the double-stranded RNA molecule described in the second aspect of the present invention.

In a fifth aspect, the present invention provides the use of compounds according to the first aspect of the invention for preparing double-stranded RNA molecules to silence target genes and suppress off-target effects of nucleic acids.

Detailed Implementation

Unless otherwise defined below, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Furthermore, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will become apparent from this specification and the accompanying drawings, and from the appended claims.

I. Definition

The term “about” when used in conjunction with a numeric value means to cover a range of numeric values that have a lower limit of 10% less than the specified numeric value and an upper limit of 10% greater than the specified numeric value.

When the term “and/or” is used to connect two or more options, it should be understood to mean any one of the options or any two or more of the options.

As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover situations consisting of the stated elements, integers, or steps.

For clarity, when referring to the sequence or sequence motif of RNAi or oligonucleotides, only the nucleotide sequence within that sequence or motif is indicated. Since the pairing of nucleoside bases A and U corresponds to the pairing of nucleoside bases A and T, in this article, for modified RNAi molecules or oligonucleotides containing a deoxynucleotide T replacing a nucleotide U, the substitution position is indicated by a U base when referring to their sequence.

In this article, for clarity, unless otherwise specified, references to RNAi or oligonucleotides should be understood to refer not only to the sequence of nucleoside bases in the nucleotide chain that makes up the molecule (i.e., the sequence) but also to any chemical modifications present thereon (if such modifications are present). Such modifications can include modifications to the phosphate backbone of the nucleotide chain, nucleoside modifications, and sugar modifications, as well as conjugations of the nucleotide chain to non-nucleoside compounds.

In this document, "target sequence" or "target gene sequence" refers to a continuous nucleotide portion of the mRNA molecule formed during the transcription of a target gene (e.g., the transthyretin (TTR) gene, hydroxyacid oxidase 1 (HAO1) gene). The target gene sequence associated with the RNAi of this invention should be at least long enough to be used as a substrate for RNAi-guided nucleic acid cleavage, thereby causing a cleavage at or near the location of that sequence in the mRNA molecule formed by the transcription of the target gene. For example, the length of the target sequence can be, for example, 15-36 nucleotides ("nt"), or any sub-length therein. As a non-limiting example, the length of the target sequence can be 15-30 nt, 15-26 nt, 15-23 nt, 15-22 nt, 15-21 nt, 15-20 nt, 15-19 nt, 15-18 nt, 15-17 nt, 18-30 nt, 18-26 nt, 18-23 nt, 18-22 nt, 18-21 nt, 18-20 nt, 18 nt, 19-30 nt, 19-26 nt, 19-23nt, 19-22nt, 19-21nt, 19-20nt, 19nt, 20-30nt, 20-26nt, 20-25nt, 20-24nt, 20-23nt, 20-22nt, 20-21nt, 20nt, 21-30nt, 21-26nt, 21-25nt, 21-24nt, 21-23nt, or 21-22nt, 21nt, 22nt, or 23nt. In some embodiments of the invention, the target sequence is preferably at least 18 nucleotides long, more preferably at least 19 nucleotides long. In some embodiments of the invention, the target sequence is about 19 to about 30 nucleotides long. In some embodiments of the invention, the target sequence is about 19 to about 25 nucleotides long. In some embodiments of the invention, the target sequence is about 19 to about 23 nucleotides long. In some embodiments of the invention, the target sequence is about 21 to about 23 nucleotides long. In the antisense strand of RNAi, the sequence region that is complementary to a series of nucleotides in the target gene sequence is also referred to as a "sequence motif" in this paper.

In this document, unless otherwise specified, the terms "complementarity" or "complementarity" refer to the ability of an oligonucleotide or polynucleotide containing a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing a second nucleotide sequence under certain conditions and form a double-stranded structure. Those skilled in the art can determine the optimal complementarity of the two sequences and the conditions used to determine this complementarity based on the final application of the hybridized oligonucleotide or polynucleotide. Therefore, in this document, when describing the base pairing between the sense and antisense strands of RNAi, or the base pairing between the antisense strand and the target sequence of RNAi, the terms "complementarity" or "complementarity" should be understood to cover not only 100% complementarity (i.e., perfect complementarity) but also cases of less than 100% complementarity, i.e., the presence of base mismatches in the complementary double-stranded nucleic acid region that do not substantially affect the RNAi's intended function. As those skilled in the art will appreciate, in double-stranded nucleic acid molecules, when a base on one strand forms a Watson-Crick base pair with a corresponding base on the other strand in a complementary manner, the bases at that position on both strands are considered to be "complementarily paired" or "matched." For example, the purine base adenine (A) is complementary to the pyrimidine base thymine (T) or uracil (U); the purine base guanine (C) is complementary to the pyrimidine base cytosine (G). Correspondingly, a "mismatch" refers to a situation in double-stranded nucleic acids where corresponding bases on one strand are not complementary to each other. However, it should be understood that nucleotides modified in the base portion of RNA nucleosides should also be considered complementary if Watson-Crick base pairing is permitted. Therefore, in this paper, nucleoside base “complementarity” encompasses Watson-Crick base pairing between unmodified and modified nucleobases (see, for example, Hirao et al. (2012) Accounts of Chemical Research, Vol. 45, p. 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry, Suppl. 37, 1.4.1).

In this document, for the purposes of this invention, the expression "complementary" or "complementarity" associated with the double-stranded RNA molecule described herein is preferably not less than 70%, meaning that at least 70% of the base positions in the double-stranded region formed by complementary hybridization are complementary, i.e., the number of mismatched positions in the continuous nucleotide sequence forming the double-stranded region is less than 30%. For example, for a 21-base-pair double-stranded region, not less than 70% complementarity means that the double-stranded region forms no more than 6, 5, 4, 3, 2, 1, or 0 mismatched base pairs during hybridization. Preferably, the presence of insertions and deletions is not allowed when calculating the complementarity % of the continuous nucleotide sequence in the double-stranded region. Accordingly, in this document, the expression associated with the dsRNA molecule, "complementary (antisense) sequence" to the target sequence, or "complementary (sense) sequence" to a portion of the antisense sequence, can be "completely complementary" or "substantially complementary." "Completely complementary" means that the two sequences have 100% complementarity. When the first sequence is referred to herein as “substantially complementary” to the second sequence, the two sequences may contain one or more, but typically no more than 30%, 20%, or 10%, mismatched base pairs in the hybridized duplex, and still retain the ability to hybridize under conditions most relevant to its final application (e.g., repressing gene expression via a RISC pathway). It should be understood here that when the two oligonucleotides of an RNAi are designed to form one or more single-stranded overhangs during hybridization, such overhangs will not be considered mismatches when determining complementarity. For example, for the purposes described herein, an RNAi comprising a 21-nucleotide sense oligonucleotide chain and a 23-nucleotide antisense oligonucleotide chain may still be considered “perfectly complementary” if the longer antisense oligonucleotide contains a 21-nucleotide sequence that is perfectly complementary to the shorter sense oligonucleotide.

In this document, the term "protruding end" is used to describe an unpaired nucleotide located at the 3' or 5' end of the double-stranded region of a double-stranded oligonucleotide. In some embodiments according to the invention, the protruding end is 1 to 4 nt long and is preferably located at the 3' end of the antisense strand of the RNAi.

In this article, the nucleosides and nucleotides that make up the nucleotide chains in a double-stranded RNA molecule can be referred to as "units" or "monomers".

The terms "modified nucleotide" and "nucleotide analogue" are used interchangeably to refer to nucleotides that are not naturally occurring, in which the base, sugar, or phosphate ester bond subunit has more than one added or substituted substituent, or the subunit as a whole has been replaced with a different chemical group. An example of an analogue with more than one substitution is a bridged nucleic acid, in which the bridging unit has been added to the sugar ring by two substitutions, typically linked to the 2' and 4' carbon atoms. "Nucleotide analogs" can be included in any nucleic acid whose affinity for a portion of the target gene transcript and/or resistance to nucleases is enhanced due to modifications (bridging groups, substituents, etc.), including: hexetol nucleic acid (HNA), cyclohexane nucleic acid (CeNA), peptide nucleic acid (PNA), diol nucleic acid (GNA), threonine nucleic acid (TNA), morpholinonucleotide, tricyclic DNA (tcDNA), 2′-O-methylated nucleic acid, 2′-O-MOE (2′-O-methoxyethyl)-modified nucleic acid, 2′-AP (2′-O-aminopropyl)-modified nucleic acid, 2′-fluorinated nucleic acid, 2′-F-arabinose nucleic acid (2′-F-ANA), and BNA (bridging nucleic acid).

In this document, "optional" or "optionally" means that the event or condition described thereafter may or may not occur, and the description includes both the possibility that the event or condition occurs and the possibility that it does not occur. For example, "alkyl" in "optionally substituted" includes "alkyl" and "substituted alkyl" as defined below. Those skilled in the art will understand that for any group containing one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is spatially impractical, synthetically infeasible, and/or inherently unstable.

In this document, "alkyl" refers to a straight-chain or branched chain having a specified number of carbon atoms, which can be from 1 to 30 carbon atoms, for example, 1 to 20 carbon atoms, 12 to 16 carbon atoms. When referring to an alkyl residue having a specific number of carbons, it is intended to cover all branched and straight-chain forms having that number of carbons, and optionally, substitutions may be made.

The term "conjugate" or "coupler" refers to a new compound formed by the covalent linking (coupling) of two or more compound molecules through bivalent or multivalent compound molecules with linking functions. Such conjugates can be represented as GalNAc-siRNA, where GalNAc can be L96 (i.e., the GalNAc3 ligand), serving as a liver-targeting delivery vector. L96 can form a conjugate by coupling with either the 3' end of the positive strand of the siRNA or the 5' end of the positive strand of the siRNA.

In this document, the terms “subject,” “individual,” and “patient” are used interchangeably and refer to vertebrates, preferably mammals, and more preferably humans. Mammals include, but are not limited to, primates (e.g., human and non-human primates), laboratory animals (e.g., rodents, such as mice and rats), farm animals (e.g., cattle, pigs, sheep, and horses), grazing animals, and pets (such as dogs and cats).

In this document, the term "treatment" of a symptom and/or disease in mammals means (i) prevention of the symptom or disease, i.e., avoidance of any symptoms of the disease or symptom; (ii) suppression of the symptom or disease, i.e., prevention of the occurrence or progression of symptoms; and/or (iii) relief of the symptom or disease, i.e., eliciting symptom resolution. This term encompasses both therapeutic and preventative treatments. Therefore, in some aspects, the dsRNA molecule according to the invention can be therapeutically administered to inhibit, reduce, alleviate, stop, or reverse the progression of a disease or symptom or its symptoms associated with target gene expression, or to stabilize the development or progression of said disease or symptom or its symptoms in a subject.

In this document, the term "prevention" refers to reducing or lowering the likelihood of a subject developing a disease or disease symptoms. Therefore, in some aspects, the dsRNA molecules of the present invention can be administered prophylactically to prevent the occurrence or recurrence of a disease or condition or its symptoms associated with target gene expression in a subject. In some aspects, the subject does not yet have, but is at risk of having, the disease or condition, or is susceptible to developing the disease or condition.

In this article, “effective amount” refers to a predetermined amount of dsRNA molecules that can elicit a desired biological or medical response in a tissue, system, animal, or human, and/or an amount that prevents, inhibits, delays, or reverses the progression of a disease state or any other adverse symptom, or otherwise improves a disease state or symptom to achieve the desired therapeutic effect.

In this document, "therapeutic effective dose" and "preventive effective dose" refer to the amount that effectively achieves the desired therapeutic or preventive outcome at the required dose and for the required duration. Therapeutic and preventive effective doses can vary depending on various factors such as the disease to be treated or prevented, the individual's age, sex, and weight. Therapeutic and preventive effective doses are amounts in which any toxic or harmful effects are less than the beneficial therapeutic/preventive effects. Compared to subjects who have not received the drug, "therapeutic effective doses" and "preventive effective doses" preferably reduce measurable parameters by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80%. The ability of the dsRNA molecules of the present invention to reduce said measurable parameters can be evaluated in in vitro or animal model systems that predict therapeutic efficacy in humans. Typically, prophylactic administration is performed in subjects before the onset of disease symptoms, or before or at an earlier stage of the disease.

II. Modified nucleosides

In recent years, oligonucleotides have become a key focus in the development of nucleic acid drugs. From the perspective of high selectivity and low toxicity towards target genes, the development of nucleic acid drugs through RNA interference (RNAi) is actively underway. Due to the instability of natural double-stranded RNA, various chemically modified nucleotides have been synthesized in existing technologies for use in double-stranded RNA (dsRNA) molecules, such as modifications to the sugar ring using 2'-O-methyl (2'-OMe), 2'-F, and S-glycol nucleic acids (GNA), as well as coupling with N-acetylgalactosamine (GalNAc). However, due to the spatial constraints of Ago2, it can only tolerate 2'-F modification of the sugar ring at the second position of the antisense strand, but the nucleic acid degradation resistance provided by 2'-F modification is relatively poor. L-threononucleotides (TNA) possess a unique four-carbon sugar ring structure and exhibit good resistance to nucleases.

This invention obtains a modified nucleoside by modifying the sugar portion of TNA, and unexpectedly, when the modified nucleoside is introduced into the seed region of double-stranded RNA, a double-stranded RNA capable of silencing the target gene and suppressing off-target effects is obtained.

Therefore, the present invention provides modified nucleosides, which are nucleoside analogs having the structure of formula (I).

in,

R ₁ is selected from halogen, alkyl, -O-alkyl, -S-alkyl and -NH-alkyl, wherein the alkyl is optionally substituted by one or more substituents selected from the following: hydroxyl, cyano, halogen, -O-alkyl, -S-alkyl, -NH ₂ , -NH-alkyl and -N(alkyl) ₂ ;

_R2 is selected from H, halogens, alkyl groups, and -O-alkyl groups;

R ₃ is selected from -O-DMTr or -CH ₂ -X-DMTr, where X is O, S or NH, and DMTr represents 4,4'-dimethoxytriphenylmethyl;

Where Base is a natural or non-natural base, or a protected natural or non-natural base.

In some embodiments, the present invention provides nucleoside analogs of formula (Ia) and formula (Ib).

_R1 , _R2 , _R3 , and Base are defined as above; and

Stereochemistry is R or S, and for unspecified chiral centers it is a combination of R and S.

The present invention also provides another modified nucleoside, which is a nucleoside analog having the structure of formula (II).

in:

R ₁ is selected from halogen, alkyl, -O-alkyl, -S-alkyl and -NH-alkyl, wherein the alkyl is optionally substituted by one or more substituents selected from the following: hydroxyl, cyano, halogen, -O-alkyl, -S-alkyl, -NH ₂ , -NH-alkyl and -N(alkyl) ₂ ;

R ₃ is selected from -O-DMTr or -CH ₂ -X-DMTr, where X is O, S or NH, and DMTr represents 4,4'-dimethoxytriphenylmethyl;

R ₄ is selected from halogens, alkyl groups, and -O-alkyl groups;

Where Base is a natural or non-natural base, or a protected natural or non-natural base.

In some embodiments, the present invention provides nucleoside analogs of formula (IIa) and formula (IIb).

_R1 , _R3 , _R4 , and Base are defined as above; and

Stereochemistry is R or S, and for unspecified chiral centers it is a combination of R and S.

III. Double-stranded RNAi (dsRNA) molecules

This invention provides a double-stranded RNA (dsRNA) molecule that mediates targeted cleavage of messenger RNA (mRNA) via the RNA-induced silencing complex (RISC) pathway, thereby inhibiting the expression of target genes. The dsRNA molecule of this invention comprises a sense strand (also called a guest strand) and an antisense strand (also called a guide strand).

The sense and antisense strands in a dsRNA molecule can each be 12-40 nucleotides long. In some embodiments, the sense and antisense strands can be 14-38 nucleotides, 16-36 nucleotides, 18-34 nucleotides, and 20-32 nucleotides long, respectively. The sense and antisense strands can be of equal or unequal length.

In some embodiments, the antisense strand is 18 to 35 nucleotides in length. In some embodiments, the antisense strand is 21-25, 19-25, 19-21, or 21-23 nucleotides in length. In some specific embodiments, the antisense strand is 23 nucleotides in length. In some embodiments, the sense strand can be 18-35 nucleotides in length. In some embodiments, the sense strand is 21-25, 19-25, 19-21, or 21-23 nucleotides in length. In some specific embodiments, the sense strand is 21 nucleotides in length.

In some embodiments, the antisense strand of the dsRNA molecule is sufficiently complementary to the target sequence to mediate RNA interference, wherein the antisense strand comprises at least one nucleoside analog of formula (I) or formula (II) of the present invention at nucleotide positions 2-8 of the 5' region (counting from the 5' end of the antisense strand). In some embodiments, the antisense strand of the dsRNA molecule is sufficiently complementary to the target sequence to mediate RNA interference, wherein the antisense strand comprises one nucleoside analog of formula (I) or formula (II) of the present invention at nucleotide position 6 of the 5' region (counting from the 5' end of the antisense strand). In some embodiments, the antisense strand of the dsRNA molecule is sufficiently complementary to the target sequence to mediate RNA interference, wherein the antisense strand comprises one nucleoside analog of formula (I) or formula (II) of the present invention at nucleotide position 7 of the 5' region (counting from the 5' end of the antisense strand). In some embodiments, the antisense strand in the dsRNA molecule is sufficiently complementary to the target sequence to mediate RNA interference, wherein the antisense strand comprises a nucleoside analog of formula (I) or formula (II) of the present invention at the 8th nucleotide position of the 5' region (counting from the 5' end of the antisense strand).

In some embodiments, the dsRNA of the present invention further comprises other modified nucleosides, including but not limited to: 2′-O-methyl modified nucleosides, nucleosides containing a 5′ thiophosphate group, terminal nucleosides linked to a cholesterol derivative or a dodecanoic acid didecanoic acid group, locked nucleosides, baseless nucleosides, 2′-deoxyribonucleosides, 2′-fluorinated nucleosides, 2′-amino-modified nucleosides, 2′-alkyl-modified nucleosides, morpholino nucleosides, non-locked nucleosides (UNA, see, for example, WO 2008/147824), aminophosphates, or nucleosides containing non-natural bases, or any combination thereof.

The oligonucleotides in a dsRNA molecule may contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least twenty or more modified nucleosides, or all nucleosides of the oligonucleotide may be modified nucleosides. For each of the multiple modified nucleosides in a dsRNA molecule, the modification is independent and does not need to be the same.

In some embodiments, the dsRNAs that repress a specific target gene contain the same sequence (i.e., the nucleoside base sequence) but different modifications in the phosphate backbone, ribose, and bases. Examples of modifications that may be mentioned, in addition to nucleoside analogs comprising formula (I) or (II) of the present invention, may further include, for example: 2'-O-methyl nucleotide modification, 2′-fluoronucleotide modification, 2′-deoxyribonucleotide modification, locked nucleotide (LNA) modification, unlocked nucleotide (UNA) modification, conformation-restricted nucleotide modification, 2'-O-methoxyethyl nucleotide modification, debased nucleotide modification, 2′-amino nucleotide modification, 2′-O-allyl-nucleotide modification, 2′-C-alkyl-nucleotide modification, 2 ′-O-alkyl nucleotides, morpholino nucleotides, aminophosphamide nucleotide modifications, nucleotide modifications with non-natural bases, tetrahydropyranonucleotide modifications, 1,5-dehydrated hexadiol nucleotide modifications, cyclohexenyl nucleotide modifications, nucleotide modifications containing thiophosphate groups, nucleotide modifications containing methylphosphate groups, nucleotide modifications containing 2'-phosphates, nucleotide modifications containing 5'-phosphates, thermostable nucleotide modifications, ethylene glycol nucleotide (GNA) modifications, and 2-O-(N-methylacetamide) nucleotide modifications; and combinations thereof.

In a specific embodiment, the present invention provides a dsRNA molecule that inhibits the expression of transthyretin (TTR) gene via RNA interference. The dsRNA molecule according to the present invention exhibits good TTR gene inhibitory activity and significantly reduced off-target effects. The dsRNA molecule according to the present invention is suitable for the prevention and treatment of TTR-related diseases, such as age-related systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), pia mater/central nervous system (CNS) amyloidosis, hyperthyroxineemia, eye diseases such as Stargardt's disease, diabetic retinopathy, age-related macular degeneration (AMD), such as dry AMD and wet AMD; metabolic disorders, such as glucose and lipid homeostasis disorders, such as insulin resistance associated with type II diabetes, and cardiovascular diseases.

In some embodiments, the dsRNA molecule of the present invention for inhibiting the TTR gene comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 consecutive nucleotides, such as 15, 16, 17, 18, 19, 20, or 21, that differ from the nucleotide sequence of 5′-AACAGUGUUCUUGCUCUAUAA-3′ (SEQ ID NO:1) by no more than 3, for example 3, 2, 1, or 0 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides, such as 15, 16, 17, 18, 19, 20, or 21, that differ from the nucleotide sequence of 5′-UUAUAGAGCAAGAACACUGUUUU-3′ (SEQ ID NO:2) by no more than 3, for example 3, 2, 1, or 0 nucleotides, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides, wherein the antisense strand comprises at least one modified nucleotide of formula (I) or formula (II) at the nucleotide positions 2–8 (e.g., 5–8) of the 5' region (counting from the 5' end); optionally, all nucleotides of the sense strand and other nucleotides of the antisense strand comprise nucleotide modifications selected from: 2′-O-methyl modification and 2′-fluorine modification and deoxynucleotide; wherein the sense strand comprises at least two 2′-fluorine modifications; wherein the antisense strand comprises at least two 2′-fluorine modifications; wherein the sense strand comprises four thiophosphate bonds; wherein the antisense strand comprises four thiophosphate bonds; optionally, wherein the sense strand is conjugated with a ligand.

In one embodiment, the sense chain comprises four 2′-fluorine modifications.

In one embodiment, the four 2′-fluorine modifications are located at positions 7 and 9-11, starting from the 5′ end of the sense chain.

In one embodiment, the antisense strand includes a nucleoside modification of formula (I) or formula (II) located at position 5, 6, 7 or 8 starting from the 5' end of the antisense strand.

In one embodiment, the antisense chain further includes four 2'-fluorine modifications.

In one embodiment, the four 2'-fluorine modifications are located at positions 2, 6, 14, and 16, starting from the 5' end of the antisense chain.

In one embodiment, the sense strand comprises two phosphate thioester nucleoside bonds between three terminal nucleotides at the 5' end and two phosphate thioester nucleoside bonds between three terminal nucleotides at the 3' end.

In one embodiment, the antisense strand comprises two phosphate thioester nucleoside bonds between three terminal nucleotides at the 5' end and two phosphate thioester nucleoside bonds between three terminal nucleotides at the 3' end.

In one embodiment, the ligand is conjugated to the 3' end of the sense strand of the dsRNA molecule.

In one embodiment, the ligand is an asialic acid glycoprotein receptor (ASGPR) ligand.

In one embodiment, the ligand is one or more N-acetylgalactosamine (GalNAc) derivatives attached via a monovalent, divalent, or trivalent branched ligand.

In a specific embodiment, the present invention provides a dsRNA molecule that inhibits the expression of the hydroxyacid oxidase 1 (HAO1) gene via RNA interference. The dsRNA molecule according to the present invention exhibits good HAO1 gene inhibitory activity and significantly reduced off-target effects. The dsRNA molecule according to the present invention is suitable for the prevention and treatment of primary hyperoxaluria type 1 (PH1). In PH1 patients, excess oxalate cannot be completely excreted by the kidneys, causing the formation and deposition of calcium oxalate crystals in the kidneys and urinary tract. Kidney damage is caused by a combination of tubular toxicity from oxalate, nephrocalcinosis, and kidney obstruction caused by stones. More than 30% of patients develop end-stage renal disease (ESRD).

In some embodiments, the dsRNA molecule of the present invention for inhibiting the HAO1 gene comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 consecutive nucleotides, such as 15, 16, 17, 18, 19, 20, or 21, that differ from the nucleotide sequence of 5′-GAAUGUGAAAGUCAUCGACAA-3′ (SEQ ID NO:3) by no more than 3, for example 3, 2, 1, or 0 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides, such as 15, 16, 17, 18, 19, 20, or 21, that differ from the nucleotide sequence of 5′-UUGUCGAUGACUUUCACAUUCUG-3′ (SEQ ID NO:4) by no more than 3, for example 3, 2, 1, or 0 nucleotides, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides, wherein the antisense strand comprises at least one modified nucleotide of formula (I) or formula (II) at the nucleotide positions 2–8 (e.g., 5–8) of the 5' region (counting from the 5' end); optionally, all nucleotides of the sense strand and other nucleotides of the antisense strand comprise nucleotide modifications selected from: 2′-O-methyl modification and 2′-fluorine modification and deoxynucleotide; wherein the sense strand comprises at least two 2′-fluorine modifications; wherein the antisense strand comprises at least two 2′-fluorine modifications; wherein the sense strand comprises four thiophosphate bonds; wherein the antisense strand comprises four thiophosphate bonds; optionally, wherein the sense strand is conjugated with a ligand.

In one embodiment, the sense chain comprises three 2′-fluorine modifications.

In one embodiment, the three 2′-fluorine modifications are located at positions 7 and 10-11, starting from the 5′ end of the sense chain.

In one embodiment, the antisense strand includes a nucleoside modification of formula (I) or formula (II) located at position 5, 6, 7 or 8 starting from the 5' end of the antisense strand.

In one embodiment, the antisense chain further includes six 2'-fluorine modifications.

In one embodiment, the six 2'-fluorine modifications are located at positions 2, 6, 8, 9, 14 and 16 starting from the 5' end of the antisense chain.

In one embodiment, the sense strand comprises two phosphate thioester nucleoside bonds between three terminal nucleotides at the 5' end and two phosphate thioester nucleoside bonds between three terminal nucleotides at the 3' end.

In one embodiment, the antisense strand comprises two phosphate thioester nucleoside bonds between three terminal nucleotides at the 5' end and two phosphate thioester nucleoside bonds between three terminal nucleotides at the 3' end.

In one embodiment, the ligand is conjugated to the 3' end of the sense strand of the dsRNA molecule.

In one embodiment, the ligand is an asialic acid glycoprotein receptor (ASGPR) ligand.

In one embodiment, the ligand is one or more N-acetylgalactosamine (GalNAc) derivatives attached via a monovalent, divalent, or trivalent branched ligand.

IV. Pharmaceutical compositions of the present invention

This invention also relates to pharmaceutical compositions comprising a double-stranded RNA molecule as described herein. Such pharmaceutical compositions may comprise an effective amount of the double-stranded RNA molecule of this invention, and a pharmaceutically or physiologically acceptable carrier. The carrier is generally selected to suit the intended mode of administration and may include features for altering, maintaining, or protecting, for example, the composition's pH, molar osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or permeability. Typically, these carriers comprise aqueous solutions or alcohol/water solutions, emulsions, or suspensions, including saline and/or buffer media.

Suitable agents included in a pharmaceutical composition include, but are not limited to, antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (e.g., borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids), fillers (e.g., mannitol or glycine), chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)), complexing agents (e.g., polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin), monosaccharides, disaccharides, and other carbohydrates (e.g., glucose, mannose, or dextrin), proteins (e.g., free serum albumin, gelatin, or immunoglobulins), colorants, flavoring agents, emulsifiers, hydrophilic polymers (e.g., polyvinylpyrrolidone), low molecular weight peptides, and salt-forming counterions (e.g., acetones). Preservatives (e.g., sodium), preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenylethanol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), polyols (e.g., glycerol, propylene glycol, or polyethylene glycol), sugar alcohols (e.g., mannitol or sorbitol), suspending agents, surfactants or wetting agents (e.g., protonylene derivatives; PEG; sorbitol esters; polysorbates, such as polysorbate 20 or polysorbate 80; Triton; tromethamine; lecithin; cholesterol or tyloxapal), stabilizing agents (e.g., sucrose or sorbitol), tension enhancers (e.g., alkali metal halides, such as sodium chloride or potassium chloride or mannitol and sorbitol), and/or pharmaceutical adjuvants. Pharmaceutical compositions can be prepared using conventional excipients known in the art for pharmaceutical products, through any of a variety of techniques (Remington’s Pharmaceutical Sciences, 21st edition, University of the Sciences in Philadelphia, Philadelphia, PA, USA (2006)).

In some embodiments, pharmaceutical compositions comprising the double-stranded RNA molecule described herein can be administered orally to a subject patient in need. When the pharmaceutical compositions are administered orally, they can be formulated as tablets, capsules, granules, powders, or syrups.

In some preferred embodiments, pharmaceutical compositions comprising the double-stranded RNA molecules described herein can be administered parenterally to a subject patient in need. When the pharmaceutical compositions are administered parenterally, they can be formulated as intravenous, intramuscular, subcutaneous, or intrathecal injections, or infusions. Parenterical administration can be performed via subcutaneous, intramuscular, or intravenous injection using a syringe, optionally a pen syringe, or a mechanically driven syringe. Alternatively, parenterical administration can be performed using an infusion pump.

When considering parenteral administration, pharmaceutical compositions are typically in the form of sterile, pyrogen-free, and parenteral-acceptable compositions. Particularly suitable solvents for parenteral injection are properly preserved sterile isotonic solutions.

The pharmaceutical composition may be in lyophilized form, such as lyophilized cake.

The parenteral or oral formulations can be prepared by conventional methods. As needed, the double-stranded RNA molecules described herein can be mixed with any conventional additives or excipients, such as binders, disintegrants, lubricants, corrosives, solubilizers, suspending agents, emulsifiers, coating agents, cyclodextrins, and/or buffers.

In some embodiments, the pharmaceutical composition is formulated as an extended-release and reservoir formulation to provide extended release of the double-stranded RNA molecules described herein. Examples of extended-release formulations for injection can be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin) in the pharmaceutical composition. In one embodiment, a controlled-release formulation is prepared using biodegradable, biocompatible polymers such as ethylene-vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.

The dosage of the double-stranded RNA molecule and pharmaceutical composition disclosed herein can be determined based on the patient's weight, age, sex, disease severity, etc. Subjects can be given therapeutic doses of the pharmaceutical composition such as 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg body weight, etc. Dosing frequency can be based on regularity, such as daily, weekly, every two weeks, every three weeks, every one month, every two months, every three months, every four months, every five months, every six months, every seven months, every eight months, every nine months, every ten months, every eleven months, annually, or longer, with repeated administration. After the initial treatment regimen, treatment can be given at a lower frequency, for example, after monthly administration for three months, administration can be continued for six months, one year, or longer. Administration of the pharmaceutical composition may reduce the level of target proteins in, for example, a patient's cells, tissues, blood, urine or other compartments by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% or more.

Attached Figure Description

Figure 1 shows the in vivo activity of the modified nucleoside sequence with reduced off-target effects in mice.

abbreviation

In describing dsRNA molecules in this invention, the following abbreviations are used, including those in the tables of the above and the following embodiments:

Explanation of the abbreviations used for materials in this invention:

Example:

Example 1: Monomer Synthesis

1.1: Synthesis of Compound 14

The following process describes the synthesis of compound 14. Compounds 2 through 14 were synthesized according to the process.

Synthesis of compound 2:

Compound 1 (30.0 g, 157.73 mmol, 1.0 eq.) (Adamas, Lot.: P2016534) (In this paper, compound 1 is also referred to as "1". Similarly, compounds 2, 3, etc. are referred to as "2", "3", etc., respectively) were dissolved in ultra-dry DCM (300 mL). Imidazole (26.8 g, 394.33 mmol, 2.5 eq.) was added to the reaction system, and the reaction system was cooled to 0 °C and stirred continuously for 30 minutes. Then, tert-butyldimethylchlorosilane (26.15 g, 173.50 mmol, 1.1 eq.) was slowly added to the reaction system. After the addition was complete, the ice bath was removed, and the reaction was gradually restored to room temperature and allowed to proceed overnight at room temperature. The reaction was monitored by TLC, and compound 1 was found to be completely reacted. Water was added to the reaction system, followed by extraction twice with ethyl acetate. The organic phases were combined and washed with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give crude compound 2 (53.2 g), which was used directly in the next step. ESI-MS: m/z 305.2 [M+H] ⁺

Synthesis of compound 3:

Crude compound 2 (53.2 g) was dissolved in DMF (500 mL), stirred until completely dissolved, and the reaction system was cooled to approximately 0 °C. 60% NaH (7.57 g, 189.28 mmol, 1.2 eq.) was slowly added in portions to the reaction mixture. After the addition was complete, the reaction system was stirred at 0 °C for 30 minutes. Benzyl bromide (32.4 g, 189.28 mmol, 1.2 eq.) was slowly added dropwise to the reaction system, maintaining the temperature at approximately 0 °C. After the addition was complete, the reaction system was brought back to room temperature and reacted overnight at room temperature. TLC analysis showed that the reaction was complete, and compound 2 had completely reacted. The reaction system was slowly poured into a saturated ammonium chloride aqueous solution at approximately 0 °C. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude compound 3 (80.2 g), which was directly used in the next reaction. ESI-MS: m/z 395.2 [M+H] ⁺

Synthesis of compound 4:

Crude compound 3 (80.2 g) was added to a 500 mL round-bottom three-necked flask, and acetic acid (400 mL) and acetic anhydride (96.62 g, 946.38 mmol, 6.0 eq.) were added to the reaction system, and the mixture was stirred until completely dissolved. The reaction mixture was cooled to 0 °C and stirred at this temperature for 30 minutes. Concentrated sulfuric acid (10 mL) was slowly added dropwise to the reaction system, maintaining the temperature between 0 and 5 °C. After the addition was complete, the reaction system was allowed to return to room temperature and stirred for 8 hours until compound 3 reacted completely. The reaction system was cooled to approximately -5 °C and neutralized with ammonia to a pH of approximately 7. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude compound 4. The crude compound 4 was subjected to column chromatography (PE/EA = 4/1) to give compound 4 (34.85 g, 95.12 mmol, overall yield of three steps: 60.31%). ^¹H NMR (400 MHz, DMSO- _d⁶ ) δ 7.39–7.27 (m, 20H), 6.07–6.01 (m, 4H), 5.50–5.40 (m, 4H), 4.81–4.65 (m, 1H), 4.58 (d, 1H), 4.32–4.18 (m, 1H), 4.02–3.82 (m, 1H), 2.09 (s, 12H), 2.14–1.97 (m, 2H). ESI-MS: m/z 367.1 [M+H] ^⁺

Synthesis of compound 5:

Compound 4 (8.0 g, 21.84 mmol, 1.00 eq.) and uracil (4.90 g, 43.68 mmol, 2.0 eq.) were added to a 500 mL three-necked round-bottom flask, and ultra-dry acetonitrile (100 mL) was added and stirred to dissolve. Then, BSA (13.33 g, 65.52 mmol, 3.0 eq.) was added. The reaction system was placed in an oil bath and heated to 80 °C, and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen protection. After the reaction was complete, the reaction system was placed in an ice-water bath at 0 °C and stirred for 30 minutes. TMSOTf (4.85 g, 21.84 mmol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition was complete, the reaction system was placed in an oil bath and slowly heated to 80 °C, and reacted overnight at this temperature. TLC and LCMS analysis showed that compound 4 reacted completely. The reaction mixture was removed from the oil bath and cooled to room temperature. The reaction was quenched by adding a saturated sodium bicarbonate aqueous solution, and the system was extracted with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give crude compound 5. Crude compound 5 was subjected to column chromatography (PE/EA = 1/4) to give compound 5 (7.8 g, 18.64 mmol, 85.3% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.31(s,1H),8.92(d,J＝21.9Hz,1H),7.39–7.21(m,5H),6.72(d,J＝21.9Hz,1H),6.13(d,J＝14.8Hz,1 H),5.43(t,J＝15.3Hz,1H),4.61–4.41(m,4H),4.15–3.80(m,2H),2.01(s,3H),1.99(s,3H).ESI-MS:m/z 419.1[M+H] ⁺

Synthesis of compound 6:

Compound 5 (7.8 g, 18.64 mmol, 1.0 eq.) was dissolved in THF (80 mL), followed by the addition of sodium methoxide (2.22 g, 41.10 mmol, 2.2 eq.). The mixture was stirred at room temperature until compound 5 was completely reacted. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude compound 6. Crude compound 6 was subjected to column chromatography (DCM/MeOH = 15/1) to give compound 6 (5.92 g, 17.71 mmol, 95.01% yield). ESI-MS: m/z 335.1 [M+H] ⁺

Synthesis of compound 7:

The dried compound 6 (5.92 g, 17.71 mmol, 1.0 eq) was dissolved in ultradry pyridine (60 mL), followed by the slow addition of DMTrCl (7.20 g, 21.25 mmol, 1.2 eq), and the reaction was stirred at room temperature. TLC analysis showed that compound 6 reacted completely. Water was added to the reaction mixture, followed by two extractions with ethyl acetate. The combined organic phases were washed with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give crude compound 7 (10.8 g). This was used directly in the next reaction. ESI-MS: m/z 635.2 [M+H] ^-

Synthesis of compound 8:

The dried crude compound 7 (10.8 g) was dissolved in ultra-dry DCM (100 mL), stirred until completely dissolved, and the reaction system was cooled to approximately 0 °C. DMAP (2.60 g, 21.25 mmol, 1.2 eq) was then added, followed by the slow dropwise addition of acetyl chloride (1.67 g, 21.25 mmol, 1.2 eq) to the reaction system, maintaining the temperature at approximately 0 °C. After the addition was complete, the reaction system was brought back to room temperature and reacted overnight at room temperature. TLC analysis showed that compound 7 reacted completely. Water was added to the reaction system, followed by two extractions with dichloromethane. The combined organic phases were washed with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain crude compound 8. Crude compound 8 was subjected to column chromatography (PE/EA = 1/1) to obtain compound 8 (8.55 g, 12.61 mmol, 2-step yield: 71.20%). ^¹H NMR (400 MHz, DMSO- _d6) was performed. )δ11.36(s,1H),9.22(d,J＝21.9Hz,1H),7.75(d,J＝21.9Hz,1H),7.55(d,J＝ 2.5Hz,1H),7.35–7.22(m,15H),6.94–6.84(m,4H),7.75(d,J＝21.9Hz,1H),5 .50(dd,J＝2.4,1.6Hz,1H),4.88–4.77(m,1H),4.69(s,2H),3.91(dd,J＝5.5 ,1.5Hz,1H),3.85–3.70(m,7H),3.40–3.26(m,1H),2.02(s,3H).ESI-MS:m/z 677.3[M+H] ^-Synthesis of compound 9:

The dried compound 8 (8.55 g, 12.61 mmol, 1.0 eq) was dissolved in ultradry DCM (90 mL), stirred until completely dissolved, and the reaction system was cooled to approximately 0 °C. Triethylsilane (2.93 g, 25.22 mmol, 2.0 eq) was then added, followed by slow dropwise addition of dichloroacetic acid (1.95 g, 15.13 mmol, 1.2 eq) to the reaction system, maintaining the temperature at approximately 0 °C. After the addition was complete, the reaction system was brought back to room temperature and reacted overnight at room temperature. TLC analysis showed that compound 8 reacted completely. Water was added to the reaction system, followed by two extractions with dichloromethane. The combined organic phases were washed with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain crude compound 9. Crude compound 9 was subjected to column chromatography (PE/EA = 1/2) to obtain compound 9 (4.03 g, 10.71 mmol, 71.20% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.25(s,1H),8.34(d,J＝10.9Hz,1H),7.42–7.15(m,5H),6.35(d,J＝10.9Hz,1H),5.83(d,J＝7.8Hz,1H),5.46(t ,J＝7.8Hz,1H),4.72–4.57(m,3H),4.11(t,J＝7.9Hz,1H),3.90(s,1H),3.74–3.39(m,2H),2.02(s,3H).ESI-MS:m/z 376.1[M+H] ⁺

Synthesis of compound 10:

Compound 9 (4.03 g, 10.71 mmol, 1.0 eq) was dissolved in a mixed solvent of acetone (100 mL) and water (50 mL), stirred until completely dissolved, and the reaction system was cooled to approximately 0 °C. Silver carbonate (1.48 g, 5.36 mmol, 0.5 eq) was then added, followed by the slow addition of a selective fluorine reagent (18.97 g, 53.55 mmol, 5.0 eq) to the reaction system, maintaining the temperature at approximately 0 °C. After the addition was complete, the reaction system was brought back to room temperature and reacted overnight at room temperature. TLC analysis showed that compound 9 reacted completely. Water was added to the reaction system, followed by two extractions with dichloromethane. The combined organic phases were washed with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain crude compound 10. Crude compound 10 was subjected to column chromatography (PE/EA = 2/5) to obtain compound 10 (2.42 g, 6.64 mmol, 62.00% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.12(s,1H),8.73(d,J＝21.7Hz,1H),7.44–6.94(m,6H),6.76(d,J＝21.7Hz,1H),5 .95(d,J＝14.4Hz,1H),5.56(dd,J＝17.2,14.3Hz,1H),4.76–4.37(m,3H),2.02(s,3H). ¹⁹ F NMR (377MHz, DMSO-d ₆ )δ-120.61(s).ESI-MS: m/z 365.1[M+H] ⁺

Synthesis of compound 11:

Compound 10 (2.42 g, 6.64 mmol, 1.0 eq.) was added to a 100 mL three-necked flask, and ultra-dry dichloromethane (30 mL) was added to the reaction flask. The reaction system was cooled to -78 °C and stirred at this temperature for 30 min. Boron trichloride (1 M in toluene, 20 mL, 19.92 mmol, 3.0 eq.) was slowly added dropwise to the reaction system. After the addition was complete, the temperature was restored to -10 °C and the reaction continued until compound 10 was completely reacted. The reaction was quenched at -78 °C with triethylamine and methanol. The reaction was restored to room temperature and water was added. The mixture was extracted with dichloromethane, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude compound 11. Crude compound 11 was subjected to column chromatography (PE/EA = 1/2) to obtain compound 11 (1.03 g, 3.76 mmol, 56.63% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.13(s,1H),7.98(d,J＝21.8Hz,1H),7.26–6.70(m,1H),6.75(d,J＝21.8Hz,1H),5. 84(d,J＝12.6Hz,1H),5.21–4.91(m,1H),4.65–4.30(m,1H),4.15(s,1H),2.02(s,3H). ¹⁹ F NMR (377MHz, DMSO-d ₆ )δ-119.89(s).ESI-MS: m/z 275.1[M+H] ⁺

Synthesis of compound 12:

Compound 11 (1.03 g, 3.76 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DCE (20 mL) was added to the flask. The mixture was stirred until completely dissolved. DMTrCl (2.55 g, 7.52 mmol, 2.0 eq.), silver nitrate (639 mg, 3.76 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (4.56 g, 18.80 mmol, 5.0 eq.) were added to the reaction mixture at room temperature and stirred until homogeneous. The reaction mixture was heated to 80 °C in an oil bath and allowed to react overnight. The reaction of compound 11 was confirmed to be complete by TLC and LCMS. The reaction mixture was brought back to room temperature, and methanol was added to quench the reaction. The mixture was diluted with ethyl acetate and filtered through diatomaceous earth to obtain a filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain crude compound 12. The crude compound 12 was subjected to column chromatography (PE/EA = 1/1) to give compound 12 (1.95 g, 3.38 mmol, 89.89% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.23(s,1H),8.57(d,J＝21.8Hz,1H),8.51–8.24(m,1H),7.34–7.22(m,9H),6.95–6.85(m,4H),6.63(d ,J＝21.8Hz,1H),6.52(d,J＝12.6Hz,1H),6.02–5.90(m,1H),4.54–4.30(m,1H),3.81(s,6H),2.02(s,3H). ¹⁹ F NMR (377MHz, DMSO-d ₆ )δ-120.09(s).ESI-MS: m/z575.2[M+H] ^-

Synthesis of compound 13:

Compound 12 (1.95 g, 3.38 mmol, 1.0 eq.) was dissolved in THF (20 mL), followed by the addition of sodium methoxide (219 mg, 4.06 mmol, 1.2 eq.). The mixture was stirred at room temperature until compound 12 was completely reacted. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give crude compound 13. Crude compound 13 was subjected to column chromatography (PE/EA = 1/2) to give compound 13 (1.72 g, 3.22 mmol, 95.26% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.23(s,1H),8.57(d,J＝21.8Hz,1H),8.51–8.24(m,1H),7.34–7.22(m,9H),6.95–6.85(m,4H),6.63(d,J＝2 1.8Hz,1H),6.52(d,J＝12.6Hz,1H),6.02–5.90(m,1H),4.54–4.30(m,1H),4.14(d,J＝8.0Hz,1H),3.81(s,6H). ¹⁹ FNMR (377MHz, DMSO-d ₆ )δ-120.32(s).ESI-MS: m/z 533.2[M+H] ^-

Synthesis of compound 14:

The dried compound 13 (1.72 g, 3.22 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry dichloromethane (20 mL) was added and stirred until completely dissolved. DIPEA (832 mg, 6.44 mmol, 2.0 eq.) and DMAP (78 mg, 0.64 mmol, 0.2 eq.) were added to the reaction mixture, and the mixture was stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under nitrogen protection. CEP-Cl (1.14 g, 4.83 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature, and the reaction was carried out at room temperature for 30–60 minutes until compound 13 was completely reacted. The reaction system was quenched by adding a saturated aqueous solution of sodium bicarbonate, and the reaction mixture was extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude compound 14. The crude compound 14 was purified by column chromatography (PE/EA = 1/1) to give compound 14 (2.01 g, 2.74 mmol, 85.09% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.34(s,1H),7.86(dd,J＝18.9,8.2Hz,1H),7.42–7.19(m,9H),6.92–6.84(m,4H),5.65–5.50(m,2H),4.2 5–4.14(m,1H),4.00–3.46(m,11H),3.35–3.30(d,J＝20Hz,3H),1.09(t,J＝6.1Hz,6H),1.01(t,J＝6.8Hz,6H). ³¹ P NMR (162MHz, DMSO-d ₆ ) δ150.93 (s), 148.82 (s). ¹⁹ F NMR (377MHz, DMSO-d ₆ )δ-119.32(s),-120.12.ESI-MS:m/z 733.3[M+H] ^-

1.2: Synthesis of Compound 19

The following process describes the synthesis of compound 19. Compound 19 was synthesized according to the process.

Synthesis of compound 15:

Compound 13 (2.0 g, 3.74 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultradry DMF (30 mL) was added and stirred to dissolve. Imidazole (1.02 g, 14.96 mmol, 4.0 eq.) was added at room temperature, and the mixture was stirred for 10 minutes. Then, TBSCl (1.13 g, 7.48 mmol, 2.0 eq.) was slowly added in portions to the reaction system, and the reaction was carried out overnight at room temperature under nitrogen protection. TLC and LCMS analysis showed that compound 13 had reacted completely, and the reaction was quenched with water. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was subjected to column chromatography (PE/EA = 1/1) to give compound 15 (2.15 g, 3.32 mmol, 88.77% yield). ESI-MS: m/z 647.3 [M+H] ^-

Synthesis of compound 16:

Compound 15 (2.15 g, 3.32 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry acetonitrile (30 mL) was added and stirred until completely dissolved. Triethylamine (672 mg, 6.64 mmol, 2.0 eq.) and DMAP (811 mg, 6.64 mmol, 2.0 eq.) were then added to the reaction mixture and stirred until homogeneous. The reaction was cooled to 0–5 °C, and compound TPSCl (2.01 g, 6.64 mmol, 2.0 eq.) was slowly added in portions. After the addition was complete, the ice bath was removed and the mixture was allowed to return to room temperature. The reaction was stirred overnight at room temperature. TLC analysis showed that compound 15 reacted completely. At room temperature, ammonia (20 mL) was added to the reaction mixture and stirred for approximately 12 hours until the intermediate was completely reacted. Saturated brine was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give crude compound 16 (8.3 g, based on 100% yield). ESI-MS: m/z 646.3 [M+H] ^-

Synthesis of compound 17:

Crude compound 16 (8.3 g) was added to a 100 mL round-bottom flask, followed by pyridine (50 mL) and stirring until completely dissolved. The reaction mixture was cooled to 0 °C, and B2Cl (933 mg, 6.64 mmol, 2.0 eq.) was slowly added dropwise while stirring at 0 °C for 1 hour until compound 16 was completely reacted. The reaction was carried out under nitrogen protection throughout. The reaction mixture was brought back to room temperature and quenched with methanol and water. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude compound 17. The crude compound was subjected to column chromatography (PE/EA = 1/1) to obtain compound 17 (1.81 g, 2.41 mmol, overall yield of 72.59% in both steps). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.27(s,1H),8.64(d,J=21.8Hz,1H),8.51–8.24(m,1H),8.04–7.95(m,2H),7.68–7.49(m,3H),7.34–7.22(m,9H),6.95–6.85(m ,4H),6.49(d,J＝21.8Hz,1H),6.52(d,J＝12.6Hz,1H),6.02–5.90(m,1H),4.54–4.30(m,1H),3.81(s,6H),0.98(s,9H),0.21(s,6H). ¹⁹ F NMR (377MHz, DMSO-d ₆ )δ-119.92(s).ESI-MS:m/z 750.3[M+H] ^-

Synthesis of compound 18:

Compound 17 (1.81 g, 2.41 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and THF (20 mL) was added and stirred until completely dissolved. Triethylamine trihydrofluoride (5.0 mL) was neutralized to alkalinity with triethylamine (17 mL) and then added to the above reaction system. The reaction was carried out overnight in a 40 °C oil bath under nitrogen protection. The reaction of compound 17 was confirmed to be complete by TLC and LCMS. Water was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water, saturated sodium bicarbonate aqueous solution, and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation to obtain the crude product. The crude product was subjected to column chromatography (PE/EA = 3/2) to give compound 18 (1.17 g, 1.83 mmol, 75.93% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.25(s,1H),8.56(d,J＝21.8Hz,1H),8.53–8.25(m,1H),8.05–7.94(m,2H),7.67–7.48(m,3H),7.35–7.21(m,9H),6.96–6.82( m,4H),6.63(d,J＝21.8Hz,1H),6.52(d,J＝12.6Hz,1H),6.03–5.91(m,1H),4.55–4.31(m,1H),4.09(d,J＝8.0Hz,1H),3.80(s,6H). ¹⁹ F NMR (377MHz, DMSO-d ₆ )δ-120.11(s).ESI-MS:m/z 636.2[M+H] ^-

Synthesis of compound 19:

The dried compound 18 (1.17 g, 1.83 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and 20 mL of ultra-dry dichloromethane was added and stirred to dissolve it. DIPEA (473 mg, 3.66 mmol, 2.0 eq.) and DMAP (45 mg, 0.37 mmol, 0.2 eq.) were then added to the reaction mixture, which was then protected under nitrogen purging. The reaction was cooled to approximately 0 °C, and CEP-Cl (651 mg, 2.75 mmol, 1.5 eq.) was slowly added dropwise. The reaction was continued at this temperature under nitrogen protection for 1 hour until compound 18 was completely reacted. After the reaction was complete, a saturated sodium bicarbonate aqueous solution was added to quench the reaction. The mixture was extracted twice with dichloromethane, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum evaporation to obtain the crude product. The crude product was purified by column chromatography (PE/EA = 1/1) to give compound 19 (1.22 g, 1.46 mmol, 79.78% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.24(s,1H),8.46(dd,J＝18.9,8.2Hz,1H),8.05–7.94(m,2H),7.67–7.48(m,3H),7.42–7.19(m,9H),6.92–6.84(m,4H),5.6 5–5.50(m,2H),4.25–4.14(m,1H),4.00–3.46(m,11H),3.35–3.30(d,J＝20Hz,3H),1.09(t,J＝6.1Hz,6H),1.01(t,J＝6.8Hz,6H). ^31P NMR (162MHz, DMSO-d ₆ )δ149.79(s),149.31(s). ¹⁹ F NMR (377MHz, DMSO-d ₆ )δ-129.99(s),-130.12(s).ESI-MS: m/z 836.2[M+H] ^-

1.3: Synthesis of Compound 29

The following process describes the synthesis of compound 29. Compound 29 was synthesized according to the process.

Synthesis of compound 20:

Compound 4 (8.0 g, 21.84 mmol, 1.0 eq.) and N6-benzoyladenine (10.45 g, 43.68 mmol, 2.0 eq.) were added to a 500 mL three-necked round-bottom flask, and ultra-dry acetonitrile (150 mL) was added and stirred to dissolve. Then, BSA (13.33 g, 65.52 mmol, 3.0 eq.) was added. The reaction mixture was heated to 80 °C in an oil bath and stirred at this temperature for 1 hour under nitrogen protection. After the reaction was complete, the mixture was placed in an ice-water bath at 0 °C and stirred for 30 minutes. TMSOTf (4.85 g, 21.84 mmol, 1.0 eq.) was slowly added dropwise to the reaction mixture. After the addition was complete, the mixture was placed in an oil bath and slowly heated to 80 °C, and reacted overnight at this temperature. TLC and LCMS analysis showed that compound 4 reacted completely. The reaction mixture was removed from the oil bath and cooled to room temperature. A saturated aqueous sodium bicarbonate solution was added to quench the reaction, and the mixture was extracted with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give crude compound 20. The crude product was subjected to column chromatography (PE/EA = 1/5) to give compound 20 (8.50 g, 15.58 mmol, 71.34% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.17(s,1H),8.35(s,1H),8.21(s,1H),7.99–7.89(m,2H),7.67–7.47(m,3H),7.39–7.21(m,5H),6.13(d,J＝1 4.8Hz,1H),5.43(t,J＝15.3Hz,1H),4.61–4.41(m,4H),4.15–3.80(m,2H),2.01(s,3H),1.99(s,3H).ESI-MS:m/z 546.2[M+H] ⁺

Synthesis of compound 21:

Compound 20 (8.5 g, 15.58 mmol, 1.0 eq.) was dissolved in THF (90 mL), followed by the addition of sodium methoxide (1.85 g, 34.28 mmol, 2.2 eq.). The mixture was stirred at room temperature until compound 20 was completely reacted. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude compound 21. Crude compound 21 was subjected to column chromatography (PE/EA = 1/100) to obtain compound 21 (6.61 g, 14.32 mmol, 91.91% yield). ESI-MS: m/z 462.2 [M+H] ⁺

Synthesis of compound 22:

The dried compound 21 (6.61 g, 14.32 mmol, 1.0 eq) was dissolved in ultradry pyridine (70 mL), followed by the slow addition of DMTrCl (5.82 g, 17.18 mmol, 1.2 eq), and the reaction was stirred at room temperature. TLC analysis showed that compound 21 reacted completely. Water was added to the reaction mixture, followed by two extractions with ethyl acetate. The combined organic phases were washed with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give crude compound 22 (10.5 g). This was used directly in the next reaction. ESI-MS: m/z 762.3 [M+H] ^-

Synthesis of compound 23:

The dried crude compound 22 (10.5 g) was dissolved in ultra-dry DCM (100 mL), stirred until completely dissolved, and the reaction system was cooled to approximately 0 °C. DMAP (2.10 g, 17.18 mmol, 1.2 eq) was then added, followed by the slow dropwise addition of acetyl chloride (1.35 g, 17.18 mmol, 1.2 eq) to the reaction system, maintaining the temperature at approximately 0 °C. After the addition was complete, the reaction system was brought back to room temperature and reacted overnight at room temperature. TLC analysis showed that compound 22 reacted completely. Water was added to the reaction system, followed by two extractions with dichloromethane. The combined organic phases were washed with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain crude compound 23. Crude compound 23 was subjected to column chromatography (PE/EA = 1/1) to obtain compound 23 (8.80 g, 10.92 mmol, 2-step yield: 76.23%). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.36(s,1H),8.35(s,1H),8.21(s,1H),7.99–7.89(m,2H),7.67–7.47( m,3H),7.35–7.22(m,14H),6.94–6.84(m,4H),7.75(d,J＝21.9Hz,1H),5.5 0(dd,J＝2.4,1.6Hz,1H),4.88–4.77(m,1H),4.69(s,2H),3.91(dd,J＝5.5, 1.5Hz,1H),3.85–3.70(m,7H),3.40–3.26(m,1H),2.02(s,3H).ESI-MS:m/z 804.3[M+H] ^-

Synthesis of compound 24:

The dried compound 23 (8.80 g, 10.92 mmol, 1.0 eq) was dissolved in ultradry DCM (90 mL), stirred until completely dissolved, and the reaction system was cooled to approximately 0 °C. Triethylsilane (2.54 g, 21.84 mmol, 2.0 eq) was then added, followed by slow dropwise addition of dichloroacetic acid (1.69 g, 13.10 mmol, 1.2 eq) to the reaction system, maintaining the temperature at approximately 0 °C. After the addition was complete, the reaction system was brought back to room temperature and reacted overnight at room temperature. TLC analysis showed that compound 23 reacted completely. Water was added to the reaction system, followed by two extractions with dichloromethane. The combined organic phases were washed with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain crude compound 24. Crude compound 24 was subjected to column chromatography (PE/EA = 1/2) to obtain compound 24 (4.13 g, 8.20 mmol, 75.20% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.36(s,1H),8.35(s,1H),8.21(s,1H),7.99–7.89(m,2H),7.67–7.47(m,3H),7.42–7.15(m,5H),5.83(d,J＝7.8Hz,1H) ,5.46(t,J＝7.8Hz,1H),4.72–4.57(m,3H),4.11(t,J＝7.9Hz,1H),3.90(s,1H),3.74–3.39(m,2H),2.02(s,3H).ESI-MS:m/z 504.2[M+H] ⁺

Synthesis of compound 25:

Compound 24 (4.13 g, 8.20 mmol, 1.0 eq) was dissolved in a mixed solvent of acetone (100 mL) and water (50 mL), stirred until completely dissolved, and the reaction system was cooled to approximately 0 °C. Silver carbonate (1.13 g, 4.10 mmol, 0.5 eq) was then added, followed by the slow addition of a selective fluoride reagent (14.52 g, 41.00 mmol, 5.0 eq) to the reaction system, maintaining the temperature at approximately 0 °C. After the addition was complete, the reaction system was brought back to room temperature and reacted overnight at room temperature. TLC analysis showed that compound 24 had completely reacted. Water was added to the reaction system, followed by two extractions with dichloromethane. The combined organic phases were washed with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain crude compound 25. Crude compound 25 was subjected to column chromatography (PE/EA = 2/7) to obtain compound 25 (2.47 g, 5.03 mmol, 61.34% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.32(s,1H),8.32(s,1H),8.20(s,1H),7.98–7.87(m,2H),7.67–7.47(m,3H),7.44–6.94( m,6H),5.95(d,J＝14.4Hz,1H),5.56(dd,J＝17.2,14.3Hz,1H),4.76–4.37(m,3H),2.02(s,3H). ¹⁹ F NMR (377MHz, DMSO-d ₆ )δ-119.92(s).ESI-MS: m/z 492.2[M+H] ⁺

Synthesis of compound 26:

Compound 25 (2.47 g, 5.03 mmol, 1.0 eq.) was added to a 100 mL three-necked flask, and ultra-dry dichloromethane (30 mL) was added to the reaction flask. The reaction system was cooled to -78 °C and stirred at this temperature for 30 min. Boron trichloride (1 M in toluene, 15 mL, 15.09 mmol, 3.0 eq.) was slowly added dropwise to the reaction system. After the addition was complete, the temperature was restored to -10 °C and the reaction continued until compound 25 was completely reacted. The reaction was quenched at -78 °C with triethylamine and methanol. The reaction was brought back to room temperature and water was added. The mixture was extracted with dichloromethane, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude compound 26. Crude compound 26 was subjected to column chromatography (PE/EA = 1/10) to obtain compound 26 (1.26 g, 3.14 mmol, 62.43% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.23(s,1H),8.32(s,1H),8.20(s,1H),7.98–7.87(m,2H),7.67–7.47(m,3H),7.26–6.70(m ,1H),,5.84(d,J＝12.6Hz,1H),5.21–4.91(m,1H),4.65–4.30(m,1H),4.15(s,1H),2.02(s,3H). ¹⁹ F NMR (377MHz, DMSO-d ₆ )δ-119.88(s).ESI-MS: m/z 402.1[M+H] ⁺

Synthesis of compound 27:

Compound 26 (1.26 g, 3.14 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DCE (20 mL) was added to the flask. The mixture was stirred until completely dissolved. DMTrCl (2.13 g, 6.28 mmol, 2.0 eq.), silver nitrate (533 mg, 3.14 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (3.81 g, 15.70 mmol, 5.0 eq.) were added to the reaction mixture at room temperature and stirred until homogeneous. The reaction mixture was heated to 80 °C in an oil bath and allowed to react overnight. The reaction of compound 26 was confirmed to be complete by TLC and LCMS. The reaction mixture was brought back to room temperature, and methanol was added to quench the reaction. The mixture was diluted with ethyl acetate and filtered through diatomaceous earth to obtain a filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain crude compound 27. The crude product was subjected to column chromatography (PE/EA = 1/3) to give compound 27 (1.96 g, 2.79 mmol, 88.85% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.21(s,1H),8.57(d,J＝21.8Hz,1H),8.51–8.21(m,3H),7.98–7.87(m,2H),7.67–7.47(m,3H),7.34–7.22(m, 9H),6.95–6.85(m,3H),6.52(d,J＝12.6Hz,1H),6.02–5.90(m,1H),4.54–4.30(m,1H),3.81(s,6H),2.02(s,3H). ¹⁹ F NMR (377MHz, DMSO-d ₆ )δ-119.92(s).ESI-MS: m/z 702.2[M+H] ^-

Synthesis of compound 28:

Compound 27 (1.96 g, 2.79 mmol, 1.0 eq.) was dissolved in THF (20 mL), followed by the addition of sodium methoxide (181 mg, 3.35 mmol, 1.2 eq.). The mixture was stirred at room temperature until compound 27 was completely reacted. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give crude compound 28. Crude compound 28 was subjected to column chromatography (PE/EA = 1/5) to give compound 28 (1.74 g, 2.63 mmol, 94.27% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.22(s,1H),8.54(d,J＝21.8Hz,1H),8.51–8.21(m,3H),7.95–7.81(m,2H),7.62–7.47(m,3H),7.34–7 .22(m,9H),6.95–6.85(m,3H),6.52(d,J＝12.6Hz,1H),6.02–5.90(m,1H),4.54–4.30(m,1H),3.81(s,6H). ¹⁹ F NMR (377MHz, DMSO-d ₆ )δ-119.91(s).ESI-MS: m/z 660.2[M+H] ^-

Synthesis of compound 29:

The dried compound 28 (1.74 g, 2.63 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and 20 mL of ultradry dichloromethane was added and stirred until completely dissolved. DIPEA (680 mg, 5.26 mmol, 2.0 eq.) and DMAP (65 mg, 0.53 mmol, 0.2 eq.) were added to the reaction mixture, and the mixture was stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under nitrogen protection. CEP-Cl (935 mg, 3.95 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature, and the reaction was carried out at room temperature for 30–60 minutes until compound 28 was completely reacted. The reaction mixture was quenched by adding a saturated aqueous solution of sodium bicarbonate, and the reaction mixture was extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude compound 29. The crude product was purified by column chromatography (PE/EA = 1/3) to give compound 29 (2.01 g, 2.33 mmol, 88.59% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.21(s,1H),8.75–8.54(m,2H),8.05(d,J＝7.3Hz,2H),7.68–7.50(m,3H),7.39–7.31(m,2H),7.29–7.15(m,7H),6.86–6.72(m,4 H),6.05(dd,J=34.0,3.2Hz,1H),4.74–4.30(m,2H),3.87–3.78(m,1H),3.79–3.46(m,10H),2.70–2.57(m,2H),1.11–0.87(m,12H). ^31P NMR (162MHz, DMSO-d ₆ )δ151.22(s),149.21(s). ¹⁹ F NMR (377MHz, DMSO-d ₆ )δ-119.93(s),119.98(s).ESI-MS: m/z 860.3[M+H] ^-

1.4: Synthesis of Compound 39

The following process describes the synthesis of compound 39. Compound 39 was synthesized according to the process.

Synthesis of compound 30:

Compound 4 (8.0 g, 21.84 mmol, 1.00 eq.) and N2-isobutyrylguanine (9.66 g, 43.68 mmol, 2.0 eq.) were added to a 500 mL three-necked round-bottom flask, and ultradry acetonitrile (150 mL) was added and stirred to dissolve. Then, BSA (13.33 g, 65.52 mmol, 3.0 eq.) was added. The reaction system was placed in an oil bath and heated to 80 °C, and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen protection. After the reaction was complete, the reaction system was placed in an ice-water bath at 0 °C and stirred for 30 minutes. TMSOTf (4.85 g, 21.84 mmol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition was complete, the reaction system was placed in an oil bath and slowly heated to 80 °C, and reacted overnight at this temperature. TLC and LCMS analysis showed that compound 4 reacted completely. The reaction mixture was removed from the oil bath and cooled to room temperature. The reaction was quenched by adding a saturated aqueous sodium bicarbonate solution, and the mixture was extracted with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give crude compound 30. The crude product was subjected to column chromatography (PE/EA = 1/6) to give compound 30 (8.8 g, 16.68 mmol, 76.37% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ12.09(s,1H),11.53(s,1H),7.91(s,1H),7.38–7.20(m,5H),5.87(d,J＝14.8Hz,1H),5.43(t,J＝15.3Hz,1H),4.6 1–4.41(m,4H),4.15–3.80(m,2H),2.84–2.56(m,1H),2.01(s,3H),1.99(s,3H),1.10(d,J＝13.0Hz,6H).ESI-MS:m/z 528.2[M+H] ⁺

Synthesis of compound 31:

Compound 30 (8.8 g, 16.68 mmol, 1.0 eq.) was dissolved in THF (90 mL), followed by the addition of sodium methoxide (1.80 g, 33.36 mmol, 2.2 eq.). The mixture was stirred at room temperature until compound 30 was completely reacted. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude compound 31. Crude compound 31 was subjected to column chromatography (PE/EA = 1/100) to obtain compound 31 (6.81 g, 15.36 mmol, 92.09% yield). ESI-MS: m/z 444.2 [M+H] ⁺

Synthesis of compound 32:

The dried compound 31 (6.81 g, 15.36 mmol, 1.0 eq) was dissolved in ultradry pyridine (70 mL), followed by the slow addition of DMTrCl (6.24 g, 18.43 mmol, 1.2 eq), and the reaction was stirred at room temperature. TLC analysis showed that compound 31 reacted completely. Water was added to the reaction mixture, followed by two extractions with ethyl acetate. The combined organic phases were washed with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give crude compound 32 (11.2 g). This was used directly in the next reaction. ESI-MS: m/z 744.3 [M+H] ^-

Synthesis of compound 33:

The dried crude compound 32 (11.2 g) was dissolved in ultra-dry DCM (110 mL), stirred until completely dissolved, and the reaction system was cooled to approximately 0 °C. DMAP (2.25 g, 18.43 mmol, 1.2 eq) was then added, followed by the slow dropwise addition of acetyl chloride (1.45 g, 18.43 mmol, 1.2 eq) to the reaction system, maintaining the temperature at approximately 0 °C. After the addition was complete, the reaction system was brought back to room temperature and reacted overnight at room temperature. TLC analysis showed that compound 32 reacted completely. Water was added to the reaction system, followed by two extractions with dichloromethane. The combined organic phases were washed with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain crude compound 33. Crude compound 33 was subjected to column chromatography (PE/EA = 1/3) to obtain compound 33 (8.38 g, 10.64 mmol, 2-step yield: 69.27%). ¹ H NMR (400MHz, DMSO-d ₆ )δ12.09(s,1H),11.53(s,1H),7.91(s,1H),7.38–7.20(m,14H),6.96–6.74(m,4H),5.87(d,J＝14.8Hz,1H),5.43(t,J＝ 15.3Hz,1H),4.61–4.41(m,4H),4.15–3.80(m,8H),2.84–2.56(m,1H),2.01(s,3H),1.10(d,J＝13.0Hz,6H).ESI-MS:m/z 804.3[M+H] ^-

Synthesis of compound 34:

The dried compound 33 (8.38 g, 10.64 mmol, 1.0 eq) was dissolved in ultra-dry DCM (90 mL), stirred until completely dissolved, and the reaction system was cooled to approximately 0 °C. Triethylsilane (2.47 g, 21.28 mmol, 2.0 eq) was then added, followed by slow dropwise addition of dichloroacetic acid (1.65 g, 12.77 mmol, 1.2 eq) to the reaction system, maintaining the temperature at approximately 0 °C. After the addition was complete, the reaction system was brought back to room temperature and reacted overnight at room temperature. TLC analysis showed that compound 33 reacted completely. Water was added to the reaction system, followed by two extractions with dichloromethane. The combined organic phases were washed with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain crude compound 34. Crude compound 34 was subjected to column chromatography (PE/EA = 1/8) to obtain compound 34 (4.25 g, 8.75 mmol, 82.24% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ12.09(s,1H),11.53(s,1H),7.91(s,1H),7.38–7.20(m,5H),5.87(d,J＝14.8Hz,1H),5.43(t,J＝15.3Hz,1H),4.61–4. 41(m,4H),4.15–3.80(m,2H),4.11(t,J＝7.9Hz,1H),2.84–2.56(m,1H),2.01(s,3H),1.10(d,J＝13.0Hz,6H).ESI-MS:m/z 486.2[M+H] ⁺

Synthesis of compound 35:

Compound 34 (4.25 g, 8.75 mmol, 1.0 eq) was dissolved in a mixed solvent of acetone (100 mL) and water (50 mL), stirred until completely dissolved, and the reaction system was cooled to approximately 0 °C. Silver carbonate (1.21 g, 4.38 mmol, 0.5 eq) was then added, followed by the slow addition of a selective fluoride reagent (15.50 g, 43.75 mmol, 5.0 eq) to the reaction system, maintaining the temperature at approximately 0 °C. After the addition was complete, the reaction system was brought back to room temperature and reacted overnight at room temperature. TLC analysis showed that compound 34 reacted completely. Water was added to the reaction system, followed by two extractions with dichloromethane. The combined organic phases were washed with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain crude compound 35. Crude compound 35 was subjected to column chromatography (PE/EA = 1/6) to obtain compound 35 (2.53 g, 5.34 mmol, 61.03% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ12.09(s,1H),11.53(s,1H),7.91(s,1H),7.38–7.20(m,5H),5.87(d,J＝14.8Hz,1H),5.43(t,J＝15.3H z,1H),4.61–4.41(m,3H),4.11(t,J＝7.9Hz,1H),2.84–2.56(m,1H),2.01(s,3H),1.10(d,J＝13.0Hz,6H). ¹⁹ F NMR (377MHz, DMSO-d ₆ )δ-120.43(s).ESI-MS: m/z 474.2[M+H] ⁺

Synthesis of compound 36:

Compound 35 (2.53 g, 5.34 mmol, 1.0 eq.) was added to a 100 mL three-necked flask, and ultra-dry dichloromethane (30 mL) was added to the reaction flask. The reaction system was cooled to -78 °C and stirred at this temperature for 30 min. Boron trichloride (1 M in toluene, 16 mL, 16.02 mmol, 3.0 eq.) was slowly added dropwise to the reaction system. After the addition was complete, the temperature was restored to -10 °C and the reaction continued until compound 35 was completely reacted. The reaction was quenched at -78 °C with triethylamine and methanol. The reaction was brought back to room temperature and water was added. The mixture was extracted with dichloromethane, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude compound 36. Crude compound 36 was subjected to column chromatography (PE/EA = 1/20) to obtain compound 36 (1.43 g, 3.73 mmol, 69.85% yield). ESI-MS: m/z 384.1 [M+H] ⁺

Synthesis of compound 37:

Compound 36 (1.43 g, 3.73 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DCE (20 mL) was added to the flask. The mixture was stirred until completely dissolved. DMTrCl (2.53 g, 7.46 mmol, 2.0 eq.), silver nitrate (634 mg, 3.73 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (4.52 g, 18.65 mmol, 5.0 eq.) were added to the reaction mixture at room temperature and stirred until homogeneous. The reaction mixture was heated to 80 °C in an oil bath and allowed to react overnight. The reaction of compound 36 was confirmed to be complete by TLC and LCMS. The reaction mixture was brought back to room temperature, and methanol was added to quench the reaction. The mixture was diluted with ethyl acetate and filtered through diatomaceous earth to obtain a filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain crude compound 37. The crude product was subjected to column chromatography (PE/EA = 1/10) to give compound 37 (2.16 g, 3.15 mmol, 84.45% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ12.09(s,1H),11.53(s,1H),7.91(s,1H),7.38–7.20(m,9H),6.96–6.74(m,4H),5.87(d,J＝14.8Hz,1H),5.43(t,J＝15. 3Hz,1H),4.61–4.41(m,1H),4.11(t,J＝7.9Hz,1H),3.81(s,6H),2.84–2.56(m,1H),2.01(s,3H),1.10(d,J＝13.0Hz,6H). ¹⁹ F NMR (377MHz, DMSO-d ₆ )δ-120.36 (s).ESI-MS: m/z 684.3[M+H] ^-

Synthesis of compound 38:

Compound 37 (2.16 g, 3.15 mmol, 1.0 eq.) was dissolved in THF (25 mL), followed by the addition of sodium methoxide (204 mg, 3.78 mmol, 1.2 eq.). The mixture was stirred at room temperature until compound 37 was completely reacted. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give crude compound 38. Crude compound 38 was subjected to column chromatography (PE/EA = 1/20) to give compound 38 (1.81 g, 2.81 mmol, 89.21% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ12.09(s,1H),11.53(s,1H),7.91(s,1H),7.38–7.20(m,9H),6.96–6.74(m,4H),5.87(d,J＝14.8Hz,1H),5.43( t,J＝15.3Hz,1H),4.61–4.41(m,1H),4.20-4.10(m,2H),3.81(s,6H),2.84–2.56(m,1H),1.10(d,J＝13.0Hz,6H). ¹⁹ F NMR (377MHz, DMSO-d ₆ )δ-120.35(s).ESI-MS: m/z 642.2[M+H] ^-

Synthesis of compound 39:

The dried compound 38 (1.81 g, 2.81 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and 20 mL of ultra-dry dichloromethane was added and stirred until completely dissolved. DIPEA (726 mg, 5.62 mmol, 2.0 eq.) and DMAP (68 mg, 0.56 mmol, 0.2 eq.) were added to the reaction mixture, and the mixture was stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under nitrogen protection. CEP-Cl (999 mg, 4.22 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature, and the reaction was carried out at room temperature for 30–60 minutes until compound 38 was completely reacted. The reaction mixture was quenched by adding a saturated aqueous solution of sodium bicarbonate, and the reaction mixture was extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude compound 39. The crude product was purified by column chromatography (PE/EA = 1/3) to give compound 39 (2.13 g, 2.52 mmol, 89.68% yield). ^¹H NMR (400 MHz, DMSO- _d⁶ ) δ 12.70 (s, 1H), 11.59 (s, 1H), 8.21 (d, J = 29.6 Hz, 1H), 7.55–7.18 (m, 9H), 6.88–6.79 (m, 4H), 5.72 (dd, J = 38.6, 4.8 Hz, 1H), 5.25–5.12 (m, 1H), 4.50 (t, J = 5.5 Hz). ,1H),3.80–3.68(m,6H),3.30(d,J＝24.0,3H),3.25–3.20(m,1H),3.12(d,J＝8.4 Hz,1H),2.78–2.65(m,1H),2.57(m,1H),2.44–2.35(m,1H),1.11–0.87(m,18H). ³¹ P NMR(162MHz, DMSO-d ₆ )δ151.35(s),148.98(s). ¹⁹ F NMR(377MHz,DMSO-d ₆ )δ-120.37(s),-120.45(s).ESI-MS: m/z 842.3[M+H] ^-

1.5: Synthesis of Compound 50

The following process describes the synthesis procedure of compound 50. Compound 50 is synthesized according to the process.

Synthesis of compound 40:

Compound 1 (30.0 g, 157.73 mmol, 1.0 eq.) was dissolved in ultra-dry DCM (300 mL). Imidazole (26.8 g, 394.33 mmol, 2.5 eq.) was added to the reaction system, and the system was cooled to 0 °C and stirred continuously for 30 minutes. Then, tert-butyldiphenylchlorosilane (47.7 g, 173.50 mmol, 1.1 eq.) was slowly added to the reaction system. After the addition was complete, the ice bath was removed, and the reaction was gradually brought to room temperature and allowed to proceed overnight at room temperature. TLC analysis showed that compound 1 reacted completely. Water was added to the reaction system, followed by extraction twice with ethyl acetate. The organic phases were combined and washed with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and slurryed to give compound 40 (52.0 g). ESI-MS: m/z 429.6 [M+H] ⁺

Synthesis of compound 41:

Compound 40 (52.0 g, 121.33 mmol, 1.0 eq.) was dissolved in DMF (500 mL), stirred until completely dissolved, and the reaction system was cooled to approximately 0 °C. 60% NaH (5.82 g, 145.60 mmol, 1.2 eq.) was slowly added dropwise to the reaction system, maintaining the temperature at approximately 0 °C. After the addition was complete, the reaction system was brought to room temperature and reacted overnight at room temperature. TLC analysis showed that the reaction was complete, and compound 40 reacted completely. The reaction system was slowly poured into a saturated ammonium chloride aqueous solution at approximately 0 °C. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude compound 41 (78.3 g), which was directly used in the next reaction step. ESI-MS: m/z 519.7 [M+H] ⁺

Synthesis of compound 42:

Crude compound 41 (78.3 g) was added to a 500 mL round-bottom flask, followed by 200 mL of THF. Tetrabutylammonium fluoride (1 M in THF, 145.5 mL, 1.2 eq.) was then added to the reaction system. After the addition was complete, the reaction system was stirred at room temperature for 2 hours. TLC analysis showed that compound 41 reacted completely. Water was added to the reaction system, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude compound 42. Crude compound 42 was then subjected to column chromatography (PE/EA = 4/1) to obtain compound 42 (45.0 g, 160.53 mmol). ESI-MS: m/z 281.3 [M+H] ⁺

Synthesis of compound 43:

Compound 42 (45.0 g, 160.53 mmol, 1.00 eq.) was added to a 500 mL round-bottom flask, and toluene (400 mL) was added to the flask and stirred to dissolve. Then, imidazole (38.25 g, 561.86 mmol, 3.5 eq.) and triphenylphosphine (50.53 g, 192.64 mmol, 1.2 eq.) were added, followed by the slow addition of iodine (48.9 g, 192.64 mmol, 1.2 eq.). After the addition was complete, the reaction system was placed in an oil bath and heated to 80 °C. The mixture was stirred for 3 hours. TLC and LCMS analysis showed that compound 42 reacted completely. The reaction mixture was removed from the oil bath and cooled to room temperature. The reaction was quenched by adding an aqueous sodium sulfite solution. The mixture was extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude compound 43. The crude product was subjected to column chromatography (PE/EA = 1/4) to give compound 43 (54.4 g, 139.41 mmol, 86.8% yield). ESI-MS: m/z 391.23 [M+H] ⁺

Synthesis of compound 44:

Compound 43 (54.4 g, 139.41 mmol, 1.0 eq.) was dissolved in MeOH (500 mL), followed by the addition of potassium carbonate (19.34 g, 139.42 mmol, 1.0 eq.) and palladium on carbon (10% W, 5.4 g.). The mixture was stirred at room temperature until compound 43 was completely reacted. The system was filtered through diatomaceous earth, concentrated, extracted twice with ethyl acetate, filtered to remove salt, and concentrated under reduced pressure to obtain crude compound 44. Crude compound 44 was subjected to column chromatography (PE/EA = 4/1) to obtain compound 44 (33.2 g, 125.61 mmol, 90.1% yield). ¹ HNMR (400MHz, DMSO-d ₆ )δ7.51–7.20(m,5H),5.81(d,J＝4.0Hz,1H),4.72–4.64(m,2H),4.49(d,J＝12.0Hz,1H),4.25–4 .16(m,1H),3.71(d,J＝3.1Hz,1H),1.38(s,3H),1.25(s,3H),1.20(d,J＝6.4Hz,3H).ESI-MS:m/z 265.31[M+H] ⁺

Synthesis of compound 45:

Compound 44 (33.2 g, 125.61 mmol, 1.0 eq) was added to a 500 mL round-bottom three-necked flask, and acetic acid (300 mL) and acetic anhydride (64.12 g, 628.05 mmol, 5.0 eq.) were added to the reaction system, and the mixture was stirred until completely dissolved. The reaction mixture was cooled to 0 °C and stirred at this temperature for 30 minutes. Concentrated sulfuric acid (10 mL) was slowly added dropwise to the reaction system, maintaining the temperature between 0 and 5 °C. After the addition was complete, the reaction system was allowed to return to room temperature and stirred for another 8 hours until compound 44 was completely reacted. The reaction system was cooled to approximately -5 °C and neutralized with ammonia to a pH of approximately 7. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude compound 45. The crude compound 45 was subjected to column chromatography (PE/EA = 4/1) to give compound 45 (21.2 g, 68.76 mmol, 54.7% yield). ESI-MS: m/z 309.33 [M+H] ⁺

Synthesis of compound 46:

Compound 45 (10.0 g, 32.43 mmol, 1.00 eq.) and uracil (7.27 g, 64.86 mmol, 2.0 eq.) were added to a 500 mL three-necked round-bottom flask, and ultra-dry acetonitrile (100 mL) was added and stirred to dissolve. Then, BSA (19.79 g, 97.29 mmol, 3.0 eq.) was added. The reaction mixture was heated to 80 °C in an oil bath and stirred at this temperature for 1 hour under nitrogen protection. After the reaction was complete, the mixture was placed in an ice-water bath at 0 °C and stirred for 30 minutes. TMSOTf (7.21 g, 32.43 mmol, 1.0 eq.) was slowly added dropwise to the reaction mixture. After the addition was complete, the mixture was placed in an oil bath and slowly heated to 80 °C, and reacted overnight. TLC and LCMS analysis showed that compound 45 reacted completely. The reaction mixture was then removed from the oil bath and cooled to room temperature. The reaction was quenched by adding a saturated sodium bicarbonate aqueous solution, and the system was extracted with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give crude compound 46. The crude product was subjected to column chromatography (PE/EA = 1/3) to give compound 46 (9.6 g, 26.64 mmol, 82.1% yield). ¹ HNMR (400MHz, DMSO-d ₆ )δ11.36(s,1H),7.46–7.21(m,6H),6.32(d,J＝8.0Hz,1H),5.90(t,J＝9.0Hz,1H),5.18(s,1H),4.77(d,J＝11.5Hz,1H ),4.58(t,J＝11.0Hz,1H),4.31–4.18(m,1H),3.94(d,J＝3.4Hz,1H),2.11(s,3H),1.33(d,J＝6.3Hz,3H).ESI-MS:m/z 361.37[M+H] ⁺

Synthesis of compound 47:

The dried compound 46 (9.6 g, 26.64 mmol, 1.0 eq) was added to a 500 mL three-necked flask, and 100 mL of ultradry dichloromethane was added to the reaction flask. The reaction system was cooled to -40 °C and stirred at this temperature for 30 min. Boron trichloride (1 M in toluene, 80 mL, 79.92 mmol, 3.0 eq.) was slowly added dropwise to the reaction system. After the addition was complete, the temperature was restored to -10 °C and the reaction continued until compound 46 was completely reacted. The reaction was quenched at -10 °C with triethylamine and methanol. The reaction was brought back to room temperature and water was added. The mixture was extracted with dichloromethane, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude compound 47. Crude compound 47 was subjected to column chromatography (PE/EA = 1/2) to obtain compound 47 (5.0 g, 18.5 mol, 69.4% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.36(s,1H),7.59(s,1H),6.32(d,J＝8.0Hz,1H),5.90(t,J＝9.0Hz,1H),5.18(s,1H),4.49(d,J＝7 .8Hz,1H),4.31–4.18(m,1H),3.94(d,J＝3.4Hz,1H),2.11(s,3H),1.33(d,J＝6.3Hz,3H).ESI-MS:m/z 271.24[M+H] ⁺

Synthesis of compound 48:

Compound 47 (5.0 g, 18.5 mmol, 1.0 eq) was added to a 100 mL round-bottom flask, and ultra-dry DCE (100 mL) was added to the flask. The mixture was stirred until completely dissolved. DMTrCl (12.54 g, 37.0 mmol, 2.0 eq.), silver nitrate (3.14 g, 18.5 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (11.21 g, 92.5 mmol, 5.0 eq.) were added to the reaction mixture at room temperature and stirred until homogeneous. The reaction mixture was heated to 80 °C in an oil bath and allowed to react overnight. The reaction of compound 47 was confirmed to be complete by TLC and LCMS. The reaction mixture was brought back to room temperature, and methanol was added to quench the reaction. The mixture was diluted with ethyl acetate and filtered through diatomaceous earth to obtain a filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain crude compound 48. The crude product was subjected to column chromatography (PE/EA = 1/1) to give compound 48 (7.3 g, 12.75 mmol, 70% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.36(s,1H),7.46(d,J＝7.5Hz,3H),7.39–7.22(m,7H),6.92(dd,J＝8.9,7.3Hz,4H),6.32(d,J＝8.0Hz,1H),5.90(t,J＝9.0 Hz,1H),5.18(s,1H),4.31–4.18(m,1H),3.94(d,J＝3.4Hz,1H),3.81(s,6H),2.11(s,3H),1.33(d,J＝6.3Hz,3H).ESI-MS:m/z 573.6[M+H] ⁺

Synthesis of compound 49:

Compound 48 (7.3 g, 12.75 mmol, 1.0 eq.) was dissolved in THF (100 mL), followed by the addition of sodium methoxide (826.51 mg, 15.3 mmol, 1.2 eq.). The mixture was stirred at room temperature until compound 48 was completely reacted. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give crude compound 49. Crude compound 49 was subjected to column chromatography (PE/EA = 1/2) to give compound 49 (6.2 g, 11.68 mmol, 91.61% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.36(s,1H),7.46(d,J＝7.5Hz,3H),7.39–7.22(m,7H),6.92(dd,J＝8.9,7.3Hz,4H),6.32(d,J＝8.0Hz,1H),5.90(t,J＝9.0Hz,1 H),5.30(d,J＝3.8Hz,1H),5.18(s,1H),4.31–4.18(m,1H),3.94(d,J＝3.4Hz,1H),3.81(s,6H),1.33(d,J＝6.3Hz,3H).ESI-MS:m/z 531.58[M+H] ⁺

Synthesis of Compound 50:

The dried compound 49 (2.0 g, 3.77 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and 20 mL of ultradry dichloromethane was added and stirred until completely dissolved. DIPEA (974.5 mg, 7.54 mmol, 2.0 eq.) and DMAP (92.12 mg, 0.754 mmol, 0.2 eq.) were added to the reaction mixture, and the mixture was stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under nitrogen protection. CEP-Cl (1.34 g, 5.66 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature, and the reaction was carried out at room temperature for 30–60 minutes until compound 49 was completely reacted. The reaction mixture was quenched by adding a saturated aqueous solution of sodium bicarbonate, and the reaction mixture was extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum evaporation to obtain the crude compound 50. The crude product was purified by column chromatography (PE/EA = 1/1) to give compound 50 (2.3 g, 3.15 mmol, 83.55% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.36(s,1H),7.46(d,J＝7.5Hz,3H),7.39–7.22(m,7H),6.92(dd,J＝8.9,7.3Hz,4H),6.32(d,J＝8.0Hz,1H),5.90(t,J＝9.0Hz,1H), 5.18(s,1H),4.31–4.18(m,1H),3.99–3.80(m,9H),2.95–2.70(m,2H),2.60–2.49(m,2H),1.33(d,J=6.3Hz,3H),1.12–0.92(m,12H). ^31P NMR (162MHz, DMSO-d ₆ )δ150.02(s),149.73(s).ESI-MS:m/z 731.8[M+H] ⁺

1.6: Synthesis of Compound 55

The following process describes the synthesis of compound 55. Compound 55 was synthesized according to the process.

Synthesis of compound 51:

Compound 49 (1.98 g, 3.74 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultradry DMF (30 mL) was added and stirred to dissolve. Imidazole (1.02 g, 14.96 mmol, 4.0 eq.) was added at room temperature and stirred for 10 minutes. Then, TBSCl (1.13 g, 7.48 mmol, 2.0 eq.) was slowly added in portions to the reaction system, and the reaction was carried out overnight at room temperature under nitrogen protection. TLC and LCMS analysis showed that compound 49 reacted completely, and the reaction was quenched with water. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was subjected to column chromatography (PE/EA = 1/1) to give compound 51 (2.15 g, 3.32 mmol, 88.77% yield). ESI-MS: m/z 645.8 [M+H] ⁺

Synthesis of compound 52:

Compound 51 (2.15 g, 3.32 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry acetonitrile (30 mL) was added and stirred until completely dissolved. Triethylamine (672 mg, 6.64 mmol, 2.0 eq.) and DMAP (811 mg, 6.64 mmol, 2.0 eq.) were added to the reaction system and stirred until homogeneous. The reaction was cooled to 0–5 °C, and compound TPSCl (2.01 g, 6.64 mmol, 2.0 eq.) was slowly added in portions. After the addition was complete, the ice bath was removed and the mixture was allowed to return to room temperature. The reaction was stirred overnight at room temperature. TLC analysis showed that compound 51 reacted completely. At room temperature, ammonia water (20 mL) was added to the reaction system and stirred for approximately 12 hours until the intermediate was completely reacted. Saturated brine was added to the reaction system, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give crude compound 52 (2.3 g, based on 100% yield). ESI-MS: m/z 644.86 [M+H] ⁺

Synthesis of compound 53:

Crude compound 52 (2.3 g) was added to a 100 mL round-bottom flask, followed by pyridine (50 mL) and stirring until completely dissolved. The reaction mixture was cooled to 0 °C, and BzCl (933 mg, 6.64 mmol, 2.0 eq.) was slowly added dropwise to the mixture while stirring at 0 °C for 1 hour until compound 52 was completely reacted. The reaction was carried out under nitrogen protection throughout. The reaction mixture was brought back to room temperature and quenched with methanol and water. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude compound 53. Crude compound 53 was subjected to column chromatography (PE/EA = 1/1) to obtain compound 53 (1.81 g, 2.41 mmol, overall yield of 2 steps: 72.59%). ESI-MS: m/z 752.93 [M+H] ⁺

Synthesis of compound 54:

Compound 53 (1.81 g, 2.41 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and THF (20 mL) was added and stirred until completely dissolved. Triethylamine trihydrofluoride (5.0 mL) was neutralized to alkalinity with triethylamine (17 mL) and then added to the above reaction system. The reaction was carried out overnight in a 40 °C oil bath under nitrogen protection. The reaction of compound 53 was confirmed to be complete by TLC and LCMS. Water was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water, saturated sodium bicarbonate aqueous solution, and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation to obtain the crude product. The crude product was subjected to column chromatography (PE/EA = 1/3) to give compound 54 (1.16 g, 1.83 mmol, 75.93% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.36(s,1H),7.46(d,J＝7.5Hz,3H),7.92–7.72(m,2H),7.69–7.46(m,3 H),7.39–7.22(m,7H),6.92(dd,J＝8.9,7.3Hz,4H),6.32(d,J＝8.0Hz,1H), 5.90(t,J＝9.0Hz,1H),5.30(d,J＝3.8Hz,1H),5.18(s,1H),4.31–4.18(m,1 H),3.94(d,J＝3.4Hz,1H),3.81(s,6H),1.33(d,J＝6.3Hz,3H).ESI-MS:m/z 634.7[M+H] ⁺

Synthesis of compound 55:

The dried compound 54 (1.16 g, 1.83 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and 20 mL of ultra-dry dichloromethane was added and stirred to dissolve it. DIPEA (473 mg, 3.66 mmol, 2.0 eq.) and DMAP (45 mg, 0.37 mmol, 0.2 eq.) were then added to the reaction mixture under nitrogen purging protection. The reaction was cooled to approximately 0 °C, and CEP-Cl (651 mg, 2.75 mmol, 1.5 eq.) was slowly added dropwise. The reaction was continued at this temperature under nitrogen protection for 1 hour until compound 54 was completely reacted. After the reaction was complete, a saturated sodium bicarbonate aqueous solution was added to quench the reaction. The mixture was extracted twice with dichloromethane, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum evaporation to obtain the crude product. The crude product was purified by column chromatography (PE/EA = 1/1) to give compound 55 (1.22 g, 1.46 mmol, 79.78% yield). ^¹H NMR (400 MHz, DMSO- _d6) )δ11.36(s,1H),7.92–7.72(m,2H),7.69–7.46(m,3H),7.41(d,J＝7.5Hz,3H ),7.39–7.22(m,7H),6.92(dd,J＝8.9,7.3Hz,4H),6.32(d,J＝8.0Hz,1H),5.9 0(t,J＝9.0Hz,1H),5.18(s,1H),4.31–4.18(m,1H),3.99–3.80(m,9H),2.95 –2.70(m,2H),2.60–2.49(m,2H),1.33(d,J＝6.3Hz,3H),1.12–0.92(m,12H). ^31P NMR(162MHz, DMSO-d ₆ )δ149.92(s),149.69(s).ESI-MS: m/z 834.9[M+H] ^-

1.7: Synthesis of Compound 60

The following process describes the synthesis of compound 60. Compound 60 is synthesized according to the process.

Synthesis of compound 56:

Compound 45 (10.0 g, 32.43 mmol, 1.00 eq.) and N6-benzoyladenine (15.52 g, 64.86 mmol, 2.0 eq.) were added to a 500 mL three-necked round-bottom flask, and ultra-dry acetonitrile (150 mL) was added and stirred to dissolve. Then, BSA (19.79 g, 97.29 mmol, 3.0 eq.) was added. The reaction mixture was heated to 80 °C in an oil bath and stirred at this temperature for 1 hour under nitrogen protection. After the reaction was complete, the mixture was placed in an ice-water bath at 0 °C and stirred for 30 minutes. TMSOTf (7.21 g, 32.43 mmol, 1.0 eq.) was slowly added dropwise to the reaction mixture. After the addition was complete, the mixture was placed in an oil bath and slowly heated to 80 °C, and reacted overnight at this temperature. TLC and LCMS analysis showed that compound 45 reacted completely. The reaction mixture was removed from the oil bath and cooled to room temperature. A saturated aqueous sodium bicarbonate solution was added to quench the reaction, and the mixture was extracted with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give crude compound 56. The crude product was subjected to column chromatography (PE/EA = 1/5) to give compound 56 (11.4 g, 23.38 mmol, 72.1% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.17(s,1H),8.35(s,1H),8.21(s,1H),8.01–7.87(m,2H),7.69–7.47(m,3H),7.46–7.21(m,5H),5.90(t,J＝9.0Hz,1H),5.18(s,1H), 4.77(d,J＝11.5Hz,1H),4.58(t,J＝11.0Hz,1H),4.31–4.18(m,1H),3.94(d,J＝3.4Hz,1H),2.11(s,3H),1.33(d,J＝6.3Hz,3H).ESI-MS:m/z 488.52[M+H] ⁺

Synthesis of compound 57:

Compound 56 (11.4 g, 23.38 mmol, 1.0 eq.) was added to a 500 mL three-necked flask, and ultradry dichloromethane (100 mL) was added to the reaction flask. The reaction system was cooled to -78 °C and stirred at this temperature for 30 min. Boron trichloride (1 M in toluene, 70 mL, 70.14 mmol, 3.0 eq.) was slowly added dropwise to the reaction system. After the addition was complete, the temperature was restored to -10 °C and the reaction continued until compound 56 was completely reacted. The reaction was quenched at -78 °C with triethylamine and methanol. The reaction was brought back to room temperature and water was added. The mixture was extracted with dichloromethane, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude compound 57. Crude compound 57 was subjected to column chromatography (PE/EA = 1/10) to obtain compound 57 (4.8 g, 12.08 mmol, 51.67% yield). ESI-MS: m/z 398.39 [M+H] ⁺

Synthesis of compound 58:

Compound 57 (4.8 g, 12.08 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask, and ultra-dry DCE (50 mL) was added to the flask. The mixture was stirred until completely dissolved. DMTrCl (8.19 g, 24.16 mmol, 2.0 eq.), silver nitrate (2.05 g, 12.08 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (7.32 g, 60.40 mmol, 5.0 eq.) were added to the reaction mixture at room temperature and stirred until homogeneous. The reaction mixture was heated to 80 °C in an oil bath and allowed to react overnight. The reaction of compound 57 was confirmed to be complete by TLC and LCMS. The reaction mixture was brought back to room temperature, and methanol was added to quench the reaction. The mixture was diluted with ethyl acetate and filtered through diatomaceous earth to obtain a filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain crude compound 58. The crude product was subjected to column chromatography (PE/EA = 1/3) to give compound 58 (6.2 g, 8.86 mmol, 73.34% yield). ESI-MS: m/z 700.7 [M+H ^]

Synthesis of compound 59:

Compound 58 (6.2 g, 8.86 mmol, 1.0 eq.) was dissolved in THF (100 mL), followed by the addition of sodium methoxide (574 mg, 10.63 mmol, 1.2 eq.). The mixture was stirred at room temperature until compound 58 was completely reacted. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give crude compound 59. Crude compound 59 was subjected to column chromatography (PE/EA = 1/5) to give compound 59 (5.2 g, 7.91 mmol, 90% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.17(s,1H),8.35(s,1H),8.21(s,1H),8.01–7.87(m,2H),7.69–7.47(m,3H),7.33–7.22(m,9H),6.94–6.82(m,4H),5.90(t,J＝9.0 Hz,1H),5.23(d,J＝3.8Hz,1H),5.18(s,1H),4.31–4.18(m,1H),3.94(d,J＝3.4Hz,1H),3.82(s,6H),1.33(d,J＝6.3Hz,3H).ESI-MS:m/z 660.2[M+H] ^-

Synthesis of compound 60:

The dried compound 59 (5.2 g, 7.91 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry dichloromethane (50 mL) was added and stirred until completely dissolved. DIPEA (2.04 g, 15.82 mmol, 2.0 eq.) and DMAP (193 mg, 1.58 mmol, 0.2 eq.) were added to the reaction mixture, and the mixture was stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under nitrogen protection. CEP-Cl (2.81 g, 11.87 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature, and the reaction was carried out at room temperature for 30–60 minutes until compound 59 was completely reacted. The reaction mixture was quenched by adding a saturated aqueous solution of sodium bicarbonate, and the reaction mixture was extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude compound 60. The crude product was purified by column chromatography (PE/EA = 1/3) to give compound 60 (5.0 g, 5.83 mmol, 74% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.17(s,1H),8.35(s,1H),8.21(s,1H),8.01–7.87(m,2H),7.69–7.47 (m,3H),7.33–7.22(m,9H),6.94–6.82(m,4H),5.90(t,J＝9.0Hz,1H),5.1 8(s,1H),4.31–4.18(m,1H),3.99–3.85(m,3H),3.82(s,6H),2.96–2.74( m,2H),2.73–2.66(m,2H),1.33(d,J＝6.3Hz,3H),1.06(d,J＝12.0Hz,12H). ³¹ P NMR (162MHz, DMSO-d ₆ )δ150.91(s),149.82(s).ESI-MS: m/z 858.9[M+H] ^-

1.8: Synthesis of Compound 65

The following process describes the synthesis of compound 65. Compound 65 was synthesized according to the process.

Synthesis of compound 61:

Compound 45 (10.0 g, 32.43 mmol, 1.00 eq.) and N2-isobutyrylguanine (14.3 g, 64.86 mmol, 2.0 eq.) were added to a 500 mL three-necked round-bottom flask, and ultra-dry acetonitrile (150 mL) was added and stirred to dissolve. Then, BSA (19.8 g, 97.29 mmol, 3.0 eq.) was added. The reaction system was heated to 80 °C in an oil bath and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen protection. After the reaction was complete, the reaction system was placed in an ice-water bath at 0 °C and stirred for 30 minutes. TMSOTf (7.21 g, 32.43 mmol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition was complete, the reaction system was placed in an oil bath and slowly heated to 80 °C, and reacted overnight at this temperature. TLC and LCMS analysis showed that compound 45 reacted completely. The reaction mixture was removed from the oil bath and cooled to room temperature. A saturated aqueous sodium bicarbonate solution was added to quench the reaction, and the mixture was extracted with ethyl acetate. The organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give crude compound 61. The crude product was subjected to column chromatography (PE/EA = 1/6) to give compound 61 (11.6 g, 24.71 mmol, 76.2% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ12.09(s,1H),11.53(s,1H),7.91(s,1H),7.46–7.21(m,5H),5.90(t,J＝9.0Hz,1H),5.18(s,1H),4.77(d,J＝11.5Hz,1H),4.58(t,J＝11.0H z,1H),4.31–4.18(m,1H),3.94(d,J＝3.4Hz,1H),2.87–2.53(m,1H),2.11(s,3H),1.33(d,J＝6.3Hz,3H),1.10(d,J＝12.9Hz,6H).ESI-MS:m/z 470.5[M+H] ⁺

Synthesis of compound 62:

Compound 61 (11.6 g, 24.71 mmol, 1.0 eq.) was added to a 500 mL three-necked flask, and ultra-dry dichloromethane (150 mL) was added to the reaction flask. The reaction system was cooled to -78 °C and stirred at this temperature for 30 minutes. Boron trichloride (1 M in toluene, 74 mL, 74.13 mmol, 3.0 eq.) was slowly added dropwise to the reaction system. After the addition was complete, the temperature was restored to -10 °C and the reaction continued until compound 61 was completely reacted. The reaction was quenched at -78 °C with triethylamine and methanol. The reaction was brought back to room temperature and water was added. The mixture was extracted with dichloromethane, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude compound 62. Crude compound 62 was subjected to column chromatography (PE/EA = 1/20) to obtain compound 62 (5.6 g, 14.76 mmol, 60% yield). ESI-MS: m/z 380.37 [M+H] ⁺

Synthesis of compound 63:

Compound 62 (5.6 g, 14.76 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DCE (50 mL) was added to the flask. The mixture was stirred until completely dissolved. DMTrCl (10.0 g, 29.52 mmol, 2.0 eq.), silver nitrate (2.51 g, 14.76 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (8.9 g, 73.8 mmol, 5.0 eq.) were added to the reaction mixture at room temperature and stirred until homogeneous. The reaction mixture was heated to 80 °C in an oil bath and allowed to react overnight. The reaction of compound 62 was confirmed to be complete by TLC and LCMS. The reaction mixture was then brought back to room temperature, and methanol was added to quench the reaction. The mixture was diluted with ethyl acetate and filtered through diatomaceous earth to obtain a filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain crude compound 63. The crude product was subjected to column chromatography (PE/EA = 1/10) to give compound 63 (7.2 g, 10.56 mmol, 71.5% yield). ESI-MS: m/z 682.7 [M+H ^]

Synthesis of compound 64:

Compound 63 (7.2 g, 10.56 mmol, 1.0 eq.) was dissolved in THF (100 mL), followed by the addition of sodium methoxide (684 mg, 12.67 mmol, 1.2 eq.). The mixture was stirred at room temperature until compound 63 was completely reacted. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give crude compound 64. Crude compound 64 was subjected to column chromatography (PE/EA = 1/20) to give compound 64 (5.3 g, 8.29 mmol, 78.5% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ12.09(s,1H),11.53(s,1H),7.91(s,1H),7.38–7.20(m,9H),6.96–6.74(m,4H),5.90(t,J＝9.0Hz,1H),5.30(d,J＝3.8Hz,1H),5.18(s, 1H),4.31–4.18(m,1H),3.94(d,J＝3.4Hz,1H),3.80(s,6H),2.87–2.53(m,1H),1.33(d,J＝6.3Hz,3H),1.10(d,J＝12.9Hz,6H).ESI-MS:m/z 640.7[M+H] ^-

Synthesis of compound 65:

The dried compound 64 (2.0 g, 3.13 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and 20 mL of ultra-dry dichloromethane was added and stirred until completely dissolved. DIPEA (809 mg, 6.26 mmol, 2.0 eq.) and DMAP (77 mg, 0.63 mmol, 0.2 eq.) were added to the reaction mixture, and the mixture was stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under nitrogen protection. CEP-Cl (1.1 g, 4.70 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature, and the reaction was continued at room temperature for 30–60 minutes until compound 64 was completely reacted. The reaction mixture was quenched by adding a saturated aqueous solution of sodium bicarbonate, and the reaction mixture was extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude compound 65. The crude product was purified by column chromatography (PE/EA = 1/3) to give compound 65 (2.0 g, 2.38 mmol, 76% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ12.09(s,1H),11.53(s,1H),7.91(s,1H),7.38–7.20(m,9H),6.96–6.74(m,4H),5.90(t,J＝9.0Hz,1H),5.30(d,J＝3.8Hz,1H),5.18(s,1H), 4.31–4.18(m,1H),4.00–3.83(m,3H),3.80(s,6H),2.93–2.53(m,5H), 1.33(d,J＝6.3Hz,2H), 1.10(d,J＝12.9Hz,6H), 1.06(d,J＝12.0Hz,12H). ^31P NMR(162MHz, DMSO-d ₆ )δ151.11(s),149.91(s).ESI-MS: m/z 840.9[M+H] ^-

1.9: Synthesis of Compound 75

The following process describes the synthesis of compound 75. Compound 75 was synthesized according to the process.

Synthesis of Compound 66

The dried compound 43 (30 g, 76.88 mmol, 1.0 eq.) was added to a 500 mL round-bottom flask, and ultra-dry DMSO (200 mL) was added and stirred until completely dissolved. DBU (23.41 g, 153.76 mmol, 2.0 eq.) was then added to the reaction mixture and stirred thoroughly. The mixture was heated to 80 °C. TLC analysis showed that compound 43 reacted completely. After cooling the reaction mixture to room temperature, 0.5% citric acid aqueous solution was added. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude compound 66. The crude compound was subjected to column chromatography (PE/EA = 5/1) to obtain compound 66 (19.16 g, 73.04 mmol, 95.00% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ7.41–7.27(m,5H),6.12(d,J＝3.3Hz,1H),4.66(d,J＝3.3Hz,1H),4.63–4.49(m,3H),4.29(s,2H),1.36(s,3H),1.32(s,3H).ESI-MS: m/z 263.1[M+H] ⁺

Synthesis of Compound 67

Compound 66 (19.16 g, 73.04 mmol, 1.0 eq.) was dissolved in a mixed solvent of DCM (80 mL), acetonitrile (80 mL), and water (120 mL). Sodium periodate (62.49 g, 292.16 mmol, 4.0 eq.) was added, followed by the slow addition of ruthenium trichloride (3.03 g, 14.61 mmol, 0.2 eq.), and the reaction was stirred at room temperature. TLC analysis showed that compound 66 reacted completely. Water was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give crude compound 67. The crude product was subjected to column chromatography (PE/EA = 4/1) to give compound 67 (13.58 g, 51.38 mmol, 70.35% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ7.47–7.25(m,5H),6.40(d,J＝3.4Hz,1H),4.93(d,J＝3.4Hz,1H),4.75(s,2H),4.31(s,1H),1.40(s,3H),1.38(s,3H).ESI-MS: m/z265.1[M+H] ⁺

Synthesis of Compound 68

Compound 67 (13.58 g, 51.38 mmol, 1.0 eq.) was dissolved in MeOH (80 mL), followed by the addition of sodium borohydride (2.33 g, 61.66 mmol, 1.2 eq.), and the reaction was stirred at room temperature. TLC analysis showed that compound 67 reacted completely. Water was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give crude compound 68. The crude product was subjected to column chromatography (PE/EA = 2/1) to give compound 68 (11.23 g, 42.17 mmol, 82.07% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ7.47–7.25(m,5H),6.63(s,1H),6.40(d,J＝3.4Hz,1H),5.42(d,J＝3.1Hz,1H),4.9 3(d,J＝3.4Hz,1H),4.75(s,2H),4.31(s,1H),1.40(s,3H),1.38(s,3H).ESI-MS:m/z 267.1[M+H] ⁺

Synthesis of Compound 69

The dried compound 68 (11.23 g, 42.17 mmol, 1.0 eq.) was dissolved in DMF (110 mL), stirred until completely dissolved, and the reaction system was cooled to approximately 0 °C. 60% NaH (2.02 g, 50.60 mmol, 1.2 eq.) was slowly added dropwise to the reaction system, maintaining the temperature at approximately 0 °C. After the addition was complete, the reaction system was brought to room temperature and reacted overnight at room temperature. TLC analysis showed that the reaction was complete, and compound 68 was completely reacted. The reaction system was slowly poured into a saturated ammonium chloride aqueous solution at approximately 0 °C. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude compound 69. The crude product was subjected to column chromatography (PE/EA = 3/1) to give compound 69 (10.65 g, 38.00 mmol, 90.12% yield). ^¹H NMR (400 MHz, DMSO- _d⁶ ) δ 7.47–7.25 (m, 5H), 6.40 (d, J = 3.4 Hz, 1H), 5.42 (d, J = 3.1 Hz, 1H), 4.93 (d, J = 3.4 Hz, 1H), 4.75 (s, 2H), 4.31 (s, 1H), 3.40 (s, 3H), 1.40 (s, 3H), 1.38 (s, 3H). ESI-MS: m/z 281.1 [M+H] ^⁺

Synthesis of Compound 70

Compound 69 (10.65 g, 38.00 mmol, 1.0 eq.) was added to a 500 mL round-bottom three-necked flask, and acetic acid (100 mL) and acetic anhydride (19.40 g, 190.00 mmol, 5.0 eq.) were added to the reaction system, and the mixture was stirred until completely dissolved. The reaction mixture was cooled to 0 °C and stirred at this temperature for 30 minutes. Concentrated sulfuric acid (4 mL) was slowly added dropwise to the reaction system, maintaining the temperature between 0 and 5 °C. After the addition was complete, the reaction system was allowed to return to room temperature and stirred for another 8 hours until compound 69 reacted completely. The reaction system was cooled to approximately -5 °C and neutralized with ammonia to a pH of approximately 7. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude compound 70. The crude compound 70 was subjected to column chromatography (PE/EA = 2/1) to give compound 70 (7.86 g, 24.23 mmol, 63.76% yield). ^¹H NMR (400 MHz, DMSO- _d⁶ ) δ 7.47–7.25 (m, 5H), 6.40 (d, J = 3.4 Hz, 1H), 5.42 (d, J = 3.1 Hz, 1H), 4.93 (d, J = 3.4 Hz, 1H), 4.75 (s, 2H), 4.31 (s, 1H), 3.40 (s, 3H), 2.19 (s, 3H), 2.02 (s, 3H). ESI-MS: m/z 325.1 [M+H] ^⁺

Synthesis of Compound 71

Compound 70 (7.86 g, 24.23 mmol, 1.00 eq.) and uracil (5.43 g, 48.46 mmol, 2.0 eq.) were added to a 500 mL three-necked round-bottom flask, and ultra-dry acetonitrile (100 mL) was added and stirred to dissolve. Then, BSA (14.79 g, 72.69 mmol, 3.0 eq.) was added. The reaction system was placed in an oil bath and heated to 80 °C, and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen protection. After the reaction was complete, the reaction system was placed in an ice-water bath at 0 °C and stirred for 30 minutes. TMSOTf (5.39 g, 24.23 mmol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition was complete, the reaction system was placed in an oil bath and slowly heated to 80 °C, and reacted overnight at this temperature. TLC and LCMS analysis showed that compound 70 reacted completely. The reaction mixture was removed from the oil bath and cooled to room temperature. The reaction was quenched by adding a saturated sodium bicarbonate aqueous solution, and the system was extracted with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give crude compound 71. The crude product was subjected to column chromatography (PE/EA = 1/4) to give compound 71 (8.0 g, 21.26 mmol, 87.74% yield). ¹ HNMR (400MHz, DMSO-d ₆ )δ11.34(s,1H),7.86(d,J＝8.2Hz,1H),7.47–7.25(m,5H),6.73(d,J＝8.2Hz,1H),6.40(d,J＝3.4Hz,1H),5. 42(d,J＝3.1Hz,1H),4.93(d,J＝3.4Hz,1H),4.75(s,2H),4.31(s,1H),3.40(s,3H),2.02(s,3H).ESI-MS:m/z 377.1[M+H] ⁺

Synthesis of Compound 72

Compound 71 (8.0 g, 21.26 mmol, 1.0 eq.) was added to a 100 mL three-necked flask, and ultra-dry dichloromethane (30 mL) was added to the reaction flask. The reaction system was cooled to -78 °C and stirred at this temperature for 30 min. Boron trichloride (1 M in toluene, 64 mL, 63.78 mmol, 3.0 eq.) was slowly added dropwise to the reaction system. After the addition was complete, the temperature was restored to -10 °C and the reaction continued until compound 71 was completely reacted. The reaction was quenched at -78 °C with triethylamine and methanol. The reaction was brought back to room temperature and water was added. The mixture was extracted with dichloromethane, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude compound 72. Crude compound 72 was subjected to column chromatography (PE/EA = 1/3) to obtain compound 72 (3.53 g, 12.33 mmol, 58.00% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.34(s,1H),7.86(d,J＝8.2Hz,1H),6.73(d,J＝8.2Hz,1H),6.40(d,J＝3.4Hz,1H),5.42(d,J＝3.1Hz ,1H),4.93(d,J＝3.4Hz,1H),4.31(s,1H),4.15(d,J＝8.0Hz,1H),3.40(s,3H),2.02(s,3H).ESI-MS:m/z 287.1[M+H] ⁺

Synthesis of compound 73:

Compound 72 (3.53 g, 12.33 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask, and ultra-dry DCE (100 mL) was added to the flask. The mixture was stirred until completely dissolved. DMTrCl (8.36 g, 24.66 mmol, 2.0 eq.), silver nitrate (2.09 g, 12.33 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (14.94 g, 61.65 mmol, 5.0 eq.) were added to the reaction mixture at room temperature and stirred until homogeneous. The reaction mixture was heated to 80 °C in an oil bath and allowed to react overnight. The reaction of compound 72 was confirmed to be complete by TLC and LCMS. The reaction mixture was brought back to room temperature, and methanol was added to quench the reaction. The mixture was diluted with ethyl acetate and filtered through diatomaceous earth to obtain a filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain crude compound 73. The crude product was subjected to column chromatography (PE/EA = 1/2) to give compound 73 (6.17 g, 10.48 mmol, 85.00% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.34(s,1H),7.86(d,J＝8.2Hz,1H),7.48–7.11(m,9H),7.00–6.77(m,4H),6.73(d,J＝8.2Hz,1H),6.40(d,J＝3.4H z,1H),5.42(d,J＝3.1Hz,1H),4.93(d,J＝3.4Hz,1H),4.31(s,1H),3.80(s,6H),3.40(s,3H),2.02(s,3H).ESI-MS:m/z 587.2[M+H] ^-

Synthesis of compound 74:

Compound 73 (6.17 g, 10.48 mmol, 1.0 eq.) was dissolved in THF (60 mL), followed by the addition of sodium methoxide (680 mg, 12.58 mmol, 1.2 eq.). The mixture was stirred at room temperature until compound 73 was completely reacted. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give crude compound 74. Crude compound 74 was subjected to column chromatography (PE/EA = 2/3) to give compound 74 (5.31 g, 9.71 mmol, 92.65% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.34(s,1H),7.86(d,J＝8.2Hz,1H),7.48–7.11(m,9H),7.00–6.77(m,4H),6.73(d,J＝8.2Hz,1H),6.40(d,J＝3.4Hz,1H ),5.42(d,J＝3.1Hz,1H),4.93(d,J＝3.4Hz,1H),4.37(d,J＝8.0Hz,1H),4.31(s,1H),3.80(s,6H),3.40(s,3H).ESI-MS:m/z 545.2[M+H] ^-

Synthesis of compound 75:

The dried compound 74 (2.0 g, 3.66 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and 20 mL of ultra-dry dichloromethane was added and stirred until completely dissolved. DIPEA (946 mg, 7.32 mmol, 2.0 eq.) and DMAP (89 mg, 0.73 mmol, 0.2 eq.) were added to the reaction mixture, and the mixture was stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under nitrogen protection. CEP-Cl (1.30 g, 4.83 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature, and the reaction was carried out at room temperature for 30–60 minutes until compound 74 was completely reacted. The reaction mixture was quenched by adding a saturated aqueous solution of sodium bicarbonate, and the reaction mixture was extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude compound 75. The crude product was purified by column chromatography (PE/EA = 1/2) to give compound 75 (2.12 g, 2.84 mmol, 77.60% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.34(s,1H),7.86(d,J＝8.2Hz,1H),7.48–7.11(m,9H),7.00–6.77(m,4 H),6.73(d,J＝8.2Hz,1H),6.40(d,J＝3.4Hz,1H),5.42(d,J＝3.1Hz,1H),4. 93(d,J＝3.4Hz,1H),4.31(s,1H),3.94-3.80(m,8H),3.40(s,3H),2.92–2. 74(m,2H),2.26–2.03(m,2H),1.09(t,J＝6.1Hz,6H),1.01(t,J＝6.8Hz,6H). ³¹ P NMR (162MHz, DMSO-d ₆ )δ151.16(s),148.55(s).ESI-MS: m/z 745.3[M+H] ^-

1.10: Synthesis of Compound 80

The following process describes the synthesis of compound 80. Compound 80 is synthesized according to the process.

Synthesis of compound 76:

Compound 74 (2.0 g, 3.66 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultradry DMF (40 mL) was added and stirred to dissolve. Imidazole (997 mg, 14.64 mmol, 4.0 eq.) was added at room temperature and stirred for 10 minutes. Then, TBSCl (1.10 g, 7.32 mmol, 2.0 eq.) was slowly added in portions to the reaction system, and the reaction was carried out overnight at room temperature under nitrogen protection. TLC and LCMS analysis showed that compound 74 reacted completely, and the reaction was quenched with water. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude compound 76. Crude compound 76 was subjected to column chromatography (PE/EA = 1/2) to obtain compound 76 (2.15 g, 3.25 mmol, 88.80% yield). ESI-MS: m/z 659.3 [M+H] ^-

Synthesis of compound 77:

Compound 76 (2.15 g, 3.25 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry acetonitrile (30 mL) was added and stirred until completely dissolved. Triethylamine (658 mg, 6.50 mmol, 2.0 eq.) and DMAP (794 mg, 6.50 mmol, 2.0 eq.) were then added to the reaction mixture and stirred until homogeneous. The reaction was cooled to 0–5 °C, and compound TPSCl (1.97 g, 6.50 mmol, 2.0 eq.) was slowly added in portions. After the addition was complete, the ice bath was removed, and the mixture was allowed to return to room temperature. The reaction was stirred overnight at room temperature. TLC analysis showed that compound 76 reacted completely. At room temperature, ammonia (20 mL) was added to the reaction mixture and stirred for approximately 12 hours until the intermediate was completely reacted. Saturated brine was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give crude compound 77 (5.2 g, based on 100% yield). ESI-MS: m/z 658.3 [M+H] ^-

Synthesis of compound 78:

Crude compound 77 (5.2 g) was added to a 100 mL round-bottom flask, followed by pyridine (50 mL) and stirring until completely dissolved. The reaction mixture was cooled to 0 °C, and BzCl (914 mg, 6.50 mmol, 2.0 eq.) was slowly added dropwise while stirring at 0 °C for 1 hour until compound 77 was completely reacted. The reaction was carried out under nitrogen protection throughout. The reaction mixture was brought back to room temperature and quenched with methanol and water. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude compound. The crude compound was subjected to column chromatography (PE/EA = 1/1) to give compound 78 (1.92 g, 2.51 mmol, overall yield of 2 steps: 68.58%). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.18(s,1H),8.06–7.94(m,2H),7.86(d,J＝8.2Hz,1H),7.69–7.49( m,3H),7.47–7.11(m,9H),7.00–6.77(m,4H),6.73(d,J＝8.2Hz,1H),6.4 0(d,J＝3.4Hz,1H),5.42(d,J＝3.1Hz,1H),4.93(d,J＝3.4Hz,1H),4.31(s,1H),3.80(s,6H),3.40(s,3H),0.98(s,9H),0.21(s,6H).ESI-MS:m/z 762.3[M+H] ^-

Synthesis of compound 79:

Compound 78 (1.92 g, 2.51 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and THF (20 mL) was added and stirred until completely dissolved. Triethylamine trihydrofluoride (5.0 mL) was neutralized to alkalinity with triethylamine (17 mL) and then added to the above reaction system. The reaction was carried out overnight in a 40 °C oil bath under nitrogen protection. The reaction of compound 78 was confirmed to be complete by TLC and LCMS. Water was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water, saturated sodium bicarbonate aqueous solution, and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude compound 79. Crude compound 79 was subjected to column chromatography (PE/EA = 3/2) to obtain compound 79 (1.21 g, 1.86 mmol, 74.10% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.18(s,1H),8.06–7.94(m,2H),7.86(d,J＝8.2Hz,1H),7.69–7.49(m,3H),7.47–7.11(m,9H),7.00–6.77(m,4H),6.73(d,J＝8.2Hz,1H),6 .40(d,J＝3.4Hz,1H),5.42(d,J＝3.1Hz,1H),4.93(d,J＝3.4Hz,1H),4.31(s,1H),4.17(d,J＝8.0Hz,1H),3.80(s,6H),3.40(s,3H).ESI-MS:m/z 648.2[M+H] ^-

Synthesis of compound 80:

The dried compound 79 (1.21 g, 1.86 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and 20 mL of ultra-dry dichloromethane was added and stirred to dissolve it. DIPEA (481 mg, 3.72 mmol, 2.0 eq.) and DMAP (45 mg, 0.37 mmol, 0.2 eq.) were added to the reaction mixture, and the reaction was carried out under nitrogen purging protection. The reaction was cooled to approximately 0 °C, and CEP-Cl (660 mg, 2.79 mmol, 1.5 eq.) was slowly added dropwise. The reaction was carried out at this temperature under nitrogen protection for 1 hour until compound 79 was completely reacted. After the reaction was complete, a saturated sodium bicarbonate aqueous solution was added to quench the reaction. The mixture was extracted twice with dichloromethane, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum evaporation to obtain the crude compound 80. The crude compound 80 was purified by column chromatography (PE/EA = 1/1) to give compound 80 (1.34 g, 1.58 mmol, 84.95% yield). ^¹H NMR (400 MHz, DMSO- _d⁶ ) δ 11.18 (s, 1H), 8.06–7.94 (m, 2H), 7.86 (d, J = 8.2 Hz, 1H), 7.69–7.49 (m, 3H), 7.47–7.11 (m, 9H), 7.00–6.77 (m, 4H), 6.73 (d, J = 8.2 Hz, 1H), 6.40 (d, J = 3.4 Hz, 1H), 5.42 ( d,J＝3.1Hz,1H),4.93(d,J＝3.4Hz,1H),4.31(s,1H),3.96-3.79(m,8H),3.40(s,3H) ,2.92–2.74(m,2H),2.70–2.45(m,2H),1.09(t,J＝6.1Hz,6H),1.01(t,J＝6.8Hz,6H). ³¹ P NMR (162MHz, DMSO-d ₆ )δ149.53(s),149.11(s).ESI-MS: m/z 848.3[M+H] ^-

1.11: Synthesis of Compound 85

The following process describes the synthesis of compound 85. Compound 85 is synthesized according to the process.

Synthesis of compound 81:

Compound 70 (8.0 g, 24.67 mmol, 1.0 eq.) and N6-benzoyladenine (11.80 g, 49.34 mmol, 2.0 eq.) were added to a 500 mL three-necked round-bottom flask, and ultra-dry acetonitrile (150 mL) was added and stirred to dissolve. Then, BSA (15.06 g, 74.01 mmol, 3.0 eq.) was added. The reaction mixture was heated to 80 °C in an oil bath and stirred at this temperature for 1 hour under nitrogen protection. After the reaction was complete, the mixture was placed in an ice-water bath at 0 °C and stirred for 30 minutes. TMSOTf (5.48 g, 24.67 mmol, 1.0 eq.) was slowly added dropwise to the reaction mixture. After the addition was complete, the mixture was placed in an oil bath and slowly heated to 80 °C, and reacted overnight at this temperature. TLC and LCMS analysis showed that compound 70 reacted completely. The reaction mixture was removed from the oil bath and cooled to room temperature. A saturated aqueous sodium bicarbonate solution was added to quench the reaction, and the mixture was extracted with ethyl acetate. The organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give crude compound 81. Crude compound 81 was subjected to column chromatography (PE/EA = 1/6) to give compound 81 (8.67 g, 17.22 mmol, 69.80% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.17(s,1H),8.35(s,1H),8.21(s,1H),7.99–7.90(m,2H),7.69–7.25(m,8H),6.41(d,J＝3.4Hz,1H),5. 45(d,J＝3.1Hz,1H),4.94(d,J＝3.4Hz,1H),4.76(s,2H),4.32(s,1H),3.41(s,3H),2.03(s,3H).ESI-MS:m/z 504.2[M+H] ⁺

Synthesis of compound 82:

The dried compound 81 (8.67 g, 17.22 mmol, 1.0 eq.) was added to a 100 mL three-necked flask, and ultra-dry dichloromethane (100 mL) was added to the reaction flask. The reaction system was cooled to -78 °C and stirred at this temperature for 30 minutes. Boron trichloride (1 M in toluene, 52 mL, 51.66 mmol, 3.0 eq.) was slowly added dropwise to the reaction system. After the addition was complete, the temperature was restored to -10 °C and the reaction continued until compound 81 was completely reacted. The reaction was quenched at -78 °C with triethylamine and methanol. The reaction was brought back to room temperature and water was added. The mixture was extracted with dichloromethane, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude compound 82. Crude compound 82 was subjected to column chromatography (PE/EA = 1/10) to obtain compound 82 (4.81 g, 11.64 mmol, 67.60% yield). ESI-MS: m/z 414.1 [M+H] ⁺

Synthesis of compound 83:

Compound 82 (4.81 g, 11.64 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask, and ultra-dry DCE (100 mL) was added to the flask. The mixture was stirred until completely dissolved. DMTrCl (7.89 g, 23.28 mmol, 2.0 eq.), silver nitrate (1.98 g, 11.64 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (14.11 g, 58.2 mmol, 5.0 eq.) were added to the reaction mixture at room temperature and stirred until homogeneous. The reaction mixture was heated to 80 °C in an oil bath and allowed to react overnight. The reaction of compound 82 was confirmed to be complete by TLC and LCMS. The reaction mixture was then brought back to room temperature, and methanol was added to quench the reaction. The mixture was diluted with ethyl acetate and filtered through diatomaceous earth to obtain a filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain crude compound 83. The crude compound 83 was subjected to column chromatography (PE/EA = 1/7) to give compound 83 (7.09 g, 9.91 mmol, 85.14% yield). ¹ HNMR (400MHz, DMSO-d ₆ )δ11.17(s,1H),8.35(s,1H),8.21(s,1H),7.99–7.90(m,2H),7.69–7.46(m,3H),7.37–7.13(m,9H),6.98–6.83(m,4H),6.31(d,J＝3 .4Hz,1H),5.45(d,J＝3.1Hz,1H),4.94(d,J＝3.4Hz,1H),4.32(s,1H),3.81(s,6H),3.61(s,3H),2.03(s,3H).ESI-MS:m/z714.3[M+H] ^-

Synthesis of compound 84:

Compound 83 (7.09 g, 9.91 mmol, 1.0 eq.) was dissolved in THF (70 mL), followed by the addition of sodium methoxide (642 mg, 11.89 mmol, 1.2 eq.). The mixture was stirred at room temperature until compound 83 was completely reacted. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give crude compound 84. Crude compound 84 was subjected to column chromatography (PE/EA = 1/8) to give compound 84 (6.01 g, 8.92 mmol, 90.01% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.17(s,1H),8.35(s,1H),8.21(s,1H),7.99–7.90(m,2H),7.69–7.46(m,3H),7.37–7.13(m,9H),6.98–6.83(m,4H),6.31(d,J＝ 3.4Hz,1H),5.45(d,J＝3.1Hz,1H),4.94(d,J＝3.4Hz,1H),4.32(s,1H),4.15(d,J＝8.0Hz,1H),3.81(s,6H),3.61(s,3H).ESI-MS:m/z 672.3[M+H] ^-

Synthesis of compound 85:

The dried compound 84 (2.00 g, 2.97 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry dichloromethane (30 mL) was added and stirred until completely dissolved. DIPEA (768 mg, 5.94 mmol, 2.0 eq.) and DMAP (72 mg, 0.59 mmol, 0.2 eq.) were added to the reaction mixture, and the mixture was stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under nitrogen protection. CEP-Cl (1.06 g, 4.46 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature, and the reaction was carried out at room temperature for 30–60 minutes until compound 84 was completely reacted. The reaction mixture was quenched by adding a saturated aqueous solution of sodium bicarbonate, and the reaction mixture was extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum evaporation to obtain the crude compound 85. The crude compound 85 was purified by column chromatography (PE/EA = 1/3) to give compound 85 (1.98 g, 2.27 mmol, 76.43% yield). ^¹H NMR (400 MHz, DMSO- _d6) )δ11.17(s,1H),8.35(s,1H),8.21(s,1H),7.99–7.90(m,2H),7.69–7.46(m ,3H),7.37–7.13(m,9H),6.98–6.83(m,4H),6.31(d,J＝3.4Hz,1H),5.45(d,J ＝3.1Hz,1H),4.94(d,J＝3.4Hz,1H),4.32(s,1H),3.81(s,6H),3.61(s,3H), 2.93–2.57(m,6H),1.11–0.87(m,12H).δ150.67(s),149.46(s).ESI-MS:m/z 872.3[M+H] ^-

1.12: Synthesis of Compound 90

The following process describes the synthesis of compound 90. Compound 90 is synthesized according to the process.

Synthesis of compound 86:

Compound 70 (8.0 g, 24.67 mmol, 1.0 eq.) and N2-isobutyrylguanine (10.91 g, 49.34 mmol, 2.0 eq.) were added to a 500 mL three-necked round-bottom flask, and ultradry acetonitrile (150 mL) was added and stirred to dissolve. Then, BSA (15.06 g, 74.01 mmol, 3.0 eq.) was added. The reaction mixture was heated to 80 °C in an oil bath and stirred at this temperature for 1 hour under nitrogen protection. After the reaction was complete, the mixture was placed in an ice-water bath at 0 °C and stirred for 30 minutes. TMSOTf (5.48 g, 24.67 mmol, 1.0 eq.) was slowly added dropwise to the reaction mixture. After the addition was complete, the mixture was placed in an oil bath and slowly heated to 80 °C, and reacted overnight at this temperature. TLC and LCMS analysis showed that compound 70 reacted completely. The reaction mixture was removed from the oil bath and cooled to room temperature. A saturated aqueous sodium bicarbonate solution was added to quench the reaction, and the mixture was extracted with ethyl acetate. The organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give crude compound 86. Crude compound 86 was subjected to column chromatography (PE/EA = 1/6) to give compound 86 (7.89 g, 16.25 mmol, 65.87% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ12.708(s,1H),11.59(s,1H),8.21(d,J＝29.6Hz,1H),7.69–7.49(m,5H),6.21(d,J＝3.4Hz,1H),5.45(d,J＝3.1Hz,1H),4.94 (d,J＝3.4Hz,1H),4.76(s,2H),4.32(s,1H),3.39(s,3H),2.78–2.59(m,1H),2.03(s,3H),1.10(d,J＝13.0Hz,6H).ESI-MS:m/z 586.2[M+H] ⁺

Synthesis of compound 87:

The dried compound 86 (7.89 g, 16.25 mmol, 1.0 eq.) was added to a 100 mL three-necked flask, and ultradry dichloromethane (100 mL) was added to the reaction flask. The reaction system was cooled to -78 °C and stirred at this temperature for 30 minutes. Boron trichloride (1 M in toluene, 49 mL, 48.75 mmol, 3.0 eq.) was slowly added dropwise to the reaction system. After the addition was complete, the temperature was restored to -10 °C and the reaction continued until compound 86 was completely reacted. The reaction was quenched at -78 °C with triethylamine and methanol. The reaction was brought back to room temperature and water was added. The mixture was extracted with dichloromethane, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude compound 87. Crude compound 87 was subjected to column chromatography (PE/EA = 1/10) to obtain compound 87 (4.38 g, 11.08 mmol, 68.18% yield). ESI-MS: m/z 396.2 [M+H] ⁺

Synthesis of compound 88:

Compound 87 (4.38 g, 11.08 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask, and ultra-dry DCE (100 mL) was added to the flask. The mixture was stirred until completely dissolved. DMTrCl (7.51 g, 22.16 mmol, 2.0 eq.), silver nitrate (1.88 g, 11.08 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (13.43 g, 55.40 mmol, 5.0 eq.) were added to the reaction mixture at room temperature and stirred until homogeneous. The reaction mixture was heated to 80 °C in an oil bath and allowed to react overnight. The reaction of compound 87 was confirmed to be complete by TLC and LCMS. The reaction mixture was brought back to room temperature, and methanol was added to quench the reaction. The mixture was diluted with ethyl acetate and filtered through diatomaceous earth to obtain a filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain crude compound 88. The crude compound 88 was subjected to column chromatography (PE/EA = 1/8) to give compound 88 (6.36 g, 9.12 mmol, 82.31% yield). ¹ HNMR (400MHz, DMSO-d ₆ )δ12.708(s,1H),11.59(s,1H),8.21(d,J＝29.6Hz,1H),7.37–7.13(m,9H),6.98–6.83(m,4H),6.21(d,J＝3.4Hz,1H),5.45(d,J＝3.1Hz, 1H),4.94(d,J＝3.4Hz,1H),4.32(s,1H),3.81(s,6H),3.39(s,3H),2.78–2.59(m,1H),2.03(s,3H),1.10(d,J＝13.0Hz,6H).ESI-MS:m/z 696.3[M+H] ^-

Synthesis of compound 89:

Compound 88 (6.36 g, 9.12 mmol, 1.0 eq.) was dissolved in THF (70 mL), followed by the addition of sodium methoxide (591 mg, 10.94 mmol, 1.2 eq.). The mixture was stirred at room temperature until compound 88 was completely reacted. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude compound 89. Crude compound 89 was subjected to column chromatography (PE/EA = 1/8) to obtain compound 89 (5.61 g, 8.56 mmol, 93.86% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ12.708(s,1H),11.59(s,1H),8.21(d,J＝29.6Hz,1H),7.37–7.13(m,9H),6.98–6.83(m,4H),6.21(d,J＝3.4Hz,1H),5.45(d,J＝3.1Hz,1H), 4.94(d,J＝3.4Hz,1H),4.32(s,1H),4.13(d,J＝8.0Hz,1H),3.81(s,6H),3.39(s,3H),2.78–2.59(m,1H),1.10(d,J＝13.0Hz,6H).ESI-MS:m/z 654.3[M+H] ^-

Synthesis of compound 90:

The dried compound 89 (2.00 g, 3.05 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry dichloromethane (30 mL) was added and stirred until completely dissolved. DIPEA (788 mg, 6.1 mmol, 2.0 eq.) and DMAP (75 mg, 0.61 mmol, 0.2 eq.) were added to the reaction mixture, and the mixture was stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under nitrogen protection. CEP-Cl (1.08 g, 4.58 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature, and the reaction was carried out at room temperature for 30–60 minutes until compound 89 was completely reacted. The reaction mixture was quenched by adding a saturated aqueous solution of sodium bicarbonate, and the reaction mixture was extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude compound 90. The crude compound 90 was purified by column chromatography (PE/EA = 1/5) to give compound 90 (1.95 g, 2.28 mmol, 74.75% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ12.708(s,1H),11.59(s,1H),8.21(d,J＝29.6Hz,1H),7.37–7.13(m,9H),6.98–6.83(m,4H),6.21(d,J＝3.4Hz,1H),5.45(d,J＝3.1Hz,1H),4 .94(d,J＝3.4Hz,1H),4.32(s,1H),3.96–3.83(m,2H),3.81(s,6H),3.3 9(s,3H),2.84–2.58(m,3H),2.35(t,J=8.6Hz,2H),1.11–0.87(m,18H). ^31P NMR(162MHz, DMSO-d ₆ )δ151.66(s),148.99(s).ESI-MS: m/z 854.4[M+H] ^-

1.13: Synthesis of Compound 97

The following process describes the synthesis of compound 97. Compound 97 was synthesized according to the process.

Synthesis of compound 91:

Compound 42 (16.6 g, 59.22 mmol, 1.0 eq.) was dissolved in DMF (170 mL) and stirred until homogeneous. The reaction mixture was cooled to 0 °C and stirred for 30 minutes. Then, NaH (2.84 g, 71.06 mmol, 1.2 eq.) was added to the reaction mixture and stirred for 30 minutes. Compound iodomethane (12.61 g, 88.83 mmol, 1.5 eq.) was slowly added dropwise to the reaction system. After the addition was complete, the reaction was allowed to return to room temperature and allowed to proceed overnight at room temperature. TLC analysis showed that compound 42 reacted completely. The reaction system was slowly poured into ice water to quench the reaction. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude compound 91. The crude product was subjected to column chromatography (PE/EA = 100/9) to obtain compound 91 (15.0 g, 50.96 mmol, 86.1% yield). ESI-MS: m/z 295.2 [M+H] ⁺

Synthesis of compound 92:

Compound 91 (15.0 g, 50.96 mmol, 1.0 eq.) was added to a 500 mL round-bottom three-necked flask, and acetic acid (136.5 mL) and acetic anhydride (26.01 g, 254.95 mmol, 5.0 eq.) were added to the reaction system, and the mixture was stirred until completely dissolved. The reaction system was cooled to 0 °C and stirred at this temperature for 30 minutes. Concentrated sulfuric acid (1.37 mL) was slowly added dropwise to the reaction system, maintaining the temperature between 0 and 5 °C. After the addition was complete, the reaction system was allowed to return to room temperature and stirred for another 8 hours until compound 91 reacted completely. The reaction system was cooled to approximately -5 °C and neutralized with ammonia to a pH of approximately 7. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude compound 92. The crude product was subjected to column chromatography (PE/EA = 10/2) to give compound 92 (15.0 g, 44.33 mmol, 87.0% yield). ESI-MS: m/z 361.1 [M+Na] ⁺

Synthesis of compound 93:

Compound 92 (15.0 g, 44.33 mmol, 1.00 eq.) and uracil (9.94 g, 88.66 mmol, 2.0 eq.) were added to a 500 mL three-necked round-bottom flask. Ultra-dry acetonitrile (160 mL) was added to the flask and stirred until dissolved. Then, BSA (27.05 g, 132.99 mmol, 3.0 eq.) was added. The reaction mixture was heated to 80 °C in an oil bath and stirred at this temperature for 1 hour under nitrogen protection. After the reaction was complete, the mixture was placed in an ice-water bath at 0 °C and stirred for 30 minutes. TMSOTf (9.85 g, 44.36 mmol, 1.0 eq.) was slowly added dropwise to the reaction mixture. After the addition was complete, the mixture was placed in an oil bath and slowly heated to 80 °C, and reacted overnight. TLC and LCMS analysis showed that compound 92 reacted completely. The reaction mixture was then removed from the oil bath and cooled to room temperature. The reaction was quenched by adding a saturated sodium bicarbonate aqueous solution. The system was extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give crude compound 93. The crude product was subjected to column chromatography (PE/EA = 5/2) to give compound 93 (15.0 g, 38.42 mmol, 86.7% yield). ESI-MS: m/z 391.1 [M+H] ⁺

Synthesis of compound 94:

Compound 93 (15.0 g, 38.42 mmol, 1.0 eq.) was added to a 500 mL three-necked flask, and ultra-dry dichloromethane (200 mL) was added to the flask. The reaction system was cooled to -15 °C and stirred at this temperature for 30 min. Boron trichloride (1 M in toluene, 115 mL, 115.26 mmol, 3.0 eq.) was slowly added dropwise to the reaction system, and the reaction was continued for 5 h until compound 93 was completely reacted. The reaction was quenched at -15 °C with triethylamine and methanol. The reaction was brought back to room temperature, and water was added. The mixture was extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude compound 94. Crude compound 94 was subjected to column chromatography (PE/EA = 2/3) to obtain compound 94 (5.0 g, 16.65 mmol, 43.3% yield). ESI-MS: m/z 301.2 [M+H] ⁺

Synthesis of Compound 95:

Compound 94 (5.0 g, 16.65 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask, and ultra-dry DCE (60 mL) was added to the flask. The mixture was stirred until completely dissolved. DMTrCl (11.28 g, 33.30 mmol, 2.0 eq.), silver nitrate (2.83 g, 16.65 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (20.18 g, 166.5 mmol, 10.0 eq.) were added to the reaction mixture at room temperature and stirred until homogeneous. The reaction mixture was heated to 80 °C in an oil bath and allowed to react overnight. The reaction of compound 94 was confirmed to be complete by TLC and LCMS. The reaction mixture was brought back to room temperature, and methanol was added to quench the reaction. The mixture was diluted with ethyl acetate and filtered through diatomaceous earth to obtain a filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain crude compound 95. The crude product was subjected to column chromatography (PE/EA = 100/35) to give compound 95 (8.0 g, 13.28 mmol, 79.7% yield). ESI-MS: m/z 603.2 [M+H] ⁺

Synthesis of compound 96:

Compound 95 (8.0 g, 13.28 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask, followed by the addition of 50 mL of 7 M sodium methoxide solution at room temperature. The reaction mixture was stirred at room temperature for 3 hours until compound 95 was completely reacted. After the reaction was complete, the reaction mixture was concentrated under reduced pressure at 40 °C to obtain crude compound 96. The crude product was subjected to column chromatography (PE/EA = 1/1) to obtain compound 96 (6.0 g, 10.71 mmol, 80.6% yield). ESI-MS: m/z 561.2 [M+H] ⁺

Synthesis of compound 97:

The dried compound 96 (2.0 g, 3.57 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and 20 mL of ultra-dry dichloromethane was added and stirred until completely dissolved. DIPEA (0.93 g, 7.14 mmol, 2.0 eq.) and DMAP (87 mg, 0.714 mmol, 0.2 eq.) were added to the reaction mixture, and the mixture was stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under nitrogen protection. CEP-Cl (1.27 g, 5.36 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature, and the reaction was continued at room temperature for 30–60 minutes until compound 96 was completely reacted. The reaction mixture was quenched by adding a saturated aqueous solution of sodium bicarbonate, and the reaction mixture was extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude compound 97. The crude product was purified by column chromatography (PE/EA = 1/1) to give compound 97 (2.1 g, 2.76 mmol, 77.3% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.34(s,1H),7.86(dd,J＝18.9,8.2Hz,1H),7.42–7.19(m,9H),6.92–6.84(m,4H),5.65–5.50(m,2H),4.25–4.14(m,1H),4.0 0–3.46(m,14H),3.35–3.30(d,J＝20Hz,3H),2.73–2.68(m,1H),2.65–2.41(m,1H),1.09(t,J＝6.1Hz,6H),1.01(t,J＝6.8Hz,6H), ^31P NMR (162MHz, DMSO-d ₆ )δ151.13,148.52.ESI-MS:m/z 761.3[M+H] ⁺

1.14: Synthesis of Compound 102

The following process describes the synthesis process of compound 102. Compound 102 is synthesized according to the process.

Synthesis of compound 98:

Compound 96 (4.0 g, 7.14 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask, and 50 mL of ultradry DMF was added and stirred to dissolve. Imidazole (1.94 g, 28.56 mmol, 4.0 eq.) was added at room temperature and stirred for 10 minutes. Then, TBSCl (2.15 g, 14.28 mmol, 2.0 eq.) was slowly added in portions to the reaction system, and the reaction was carried out overnight at room temperature under nitrogen protection. TLC and LCMS analysis showed that compound 96 reacted completely, and the reaction was quenched with water. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was subjected to column chromatography (PE/EA = 2/1) to give compound 98 (3.88 g, 5.75 mmol, 80.5% yield). ESI-MS: m/z 675.3 [M+H] ⁺

Synthesis of Compound 99:

Compound 98 (3.88 g, 5.75 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask, and ultra-dry acetonitrile (30 mL) was added and stirred until completely dissolved. Triethylamine (1.16 g, 11.52 mmol, 2.0 eq.) and DMAP (1.40 g, 11.50 mmol, 2.0 eq.) were then added to the reaction mixture and stirred until homogeneous. The reaction was cooled to 0–5 °C, and compound TPSCl (3.48 g, 11.50 mmol, 2.0 eq.) was slowly added in portions. After the addition was complete, the ice bath was removed, and the mixture was allowed to return to room temperature. The reaction was stirred overnight at room temperature. TLC analysis showed that compound 98 reacted completely. At room temperature, ammonia (40 mL) was added to the reaction mixture and stirred for approximately 12 hours until the intermediate was completely reacted. Saturated brine was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give crude compound 99 (5.1 g, calculated as 100% yield). ESI-MS: m/z 674.3 [M+H] ⁺

Synthesis of Compound 100:

Crude compound 99 (5.1 g, 1.0 eq.) was added to a 100 mL round-bottom flask, followed by pyridine (50 mL) and stirring until completely dissolved. The reaction mixture was cooled to 0 °C, and BzCl (1.62 g, 11.50 mmol, 2.0 eq.) was slowly added dropwise while stirring at 0 °C for 1 hour until compound 99 was completely reacted. The reaction was carried out under nitrogen protection throughout. The reaction mixture was brought to room temperature and quenched with methanol and water. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude compound 100. The crude compound was subjected to column chromatography (PE/EA = 1/1) to obtain compound 100 (3.7 g, 4.76 mmol, overall yield of 2 steps: 82.8%). ESI-MS: m/z 778.4 [M+H] ⁺

Synthesis of compound 101:

Compound 100 (3.7 g, 4.76 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and THF (40 mL) was added and stirred until completely dissolved. Triethylamine trihydrofluoride (5.0 mL) was neutralized to alkalinity with triethylamine (17 mL) and then added to the above reaction system. The reaction was carried out overnight in a 40 °C oil bath under nitrogen protection. TLC and LCMS analysis showed that compound 100 reacted completely. Water was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water, saturated sodium bicarbonate solution, and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was subjected to column chromatography (PE/EA = 1/1) to give compound 101 (2.6 g, 3.92 mmol, 82.4% yield). ESI-MS: m/z 664.3 [M+H] ⁺

Synthesis of compound 102:

Dry compound 101 (2.6 g, 3.92 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry dichloromethane (30 mL) was added and stirred to dissolve. DIPEA (1.0 g, 7.84 mmol, 2.0 eq.) and DMAP (96 mg, 0.784 mmol, 0.2 eq.) were added to the reaction mixture, and the reaction was carried out under nitrogen purging protection. The reaction was cooled to approximately 0 °C, and CEP-Cl (1.39 g, 5.88 mmol, 1.5 eq.) was slowly added dropwise. The reaction was carried out at this temperature under nitrogen protection for 1 hour until compound 101 was completely reacted. After the reaction was complete, a saturated sodium bicarbonate aqueous solution was added to quench the reaction. The mixture was extracted twice with dichloromethane, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum evaporation to obtain the crude product. The crude product was purified by column chromatography (PE/EA = 1/1) to give compound 102 (2.3 g, 2.66 mmol, 67.9% yield). ^¹H NMR (400 MHz, DMSO- _d6) )δ11.18(d,J＝24.4Hz,1H),8.49(dd,J＝30.5,7.3Hz,1H),7.99(d,J＝7.7Hz,2 H),7.65–7.08(m,14H),6.85–6.75(m,4H),5.71–5.62(m,1H),4.22–3.82(m, 3H),3.82–3.69(m,8H),3.61–3.39(m,5H),3.35–3.30(d,J＝20Hz,3H),2.75– 2.70(m,1H),2.53–2.31(m,1H),1.08(t,J＝6.1Hz,6H),1.00(t,J＝6.8Hz,6H), ^31P NMR (162MHz, DMSO-d ₆ )δ149.52,149.10, ESI-MS: m/z 864.4[M+H] ⁺

1.15: Synthesis of Compound 107

The following process describes the synthesis process of compound 107. Compound 107 was synthesized according to the process.

Synthesis of compound 103:

Compound 92 (15.0 g, 0.04 mol, 1.00 eq.) was added to a 500 mL three-necked round-bottom flask, and ultra-dry acetonitrile (150 mL) was added and stirred to dissolve. Then, BSA (24.4 g, 0.12 mol, 3.0 eq.) and ABz (19.14 g, 0.08 mol, 2.0 eq.) were added. The reaction system was placed in an oil bath and heated to 80 °C, and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen protection. After the reaction was complete, the reaction system was placed in an ice-water bath at 0 °C and stirred for 30 minutes. TMSOTf (8.9 g, 0.04 mol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition was complete, the reaction system was placed in an oil bath and slowly heated to 80 °C, and reacted overnight at this temperature. TLC and LCMS analysis showed that compound 92 reacted completely. The reaction mixture was removed from the oil bath and cooled to room temperature. The reaction was quenched by adding a saturated sodium bicarbonate aqueous solution. The system was extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give crude compound 103. The crude product was subjected to column chromatography (PE/EA = 1/1) to give compound 103 (20.0 g, 38.64 mmol, 87.2% yield). ESI-MS: m/z 518.2 [M+H] ⁺

Synthesis of compound 104:

Compound 103 (20.0 g, 38.64 mmol, 1.0 eq.) was added to a 500 mL three-necked flask, and ultra-dry dichloromethane (200 mL) was added to the reaction flask. The reaction system was cooled to -10 °C and stirred at this temperature for 30 min. A 1.0 mol/L boron trichloride solution in dichloromethane (116 mL, 115.92 mmol, 3.0 eq.) was slowly added dropwise to the reaction system, and the reaction was continued for 5 h until compound 103 was completely reacted. The reaction was quenched at -10 °C with triethylamine and methanol. The reaction was brought back to room temperature, and water was added. The mixture was extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude compound 104. Crude compound 104 was subjected to column chromatography (PE/EA = 1/2) to obtain compound 104 (9.7 g, 22.69 mmol, 58.7% yield). ESI-MS: m/z 428.2 [M+H] ⁺

Synthesis of compound 105:

Compound 104 (9.7 g, 22.69 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DCE (100 mL) was added to the flask. The mixture was stirred until completely dissolved. DMTrCl (15.4 g, 45.38 mmol, 2.0 eq.), silver nitrate (3.85 g, 22.69 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (27.50 g, 226.9 mmol, 10.0 eq.) were added to the reaction mixture at room temperature and stirred until homogeneous. The reaction mixture was heated to 80 °C in an oil bath and allowed to react overnight. The reaction of compound 104 was confirmed to be complete by TLC and LCMS. The reaction mixture was brought back to room temperature, and methanol was added to quench the reaction. The mixture was diluted with ethyl acetate and filtered through diatomaceous earth to obtain a filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain crude compound 105. The crude product was subjected to column chromatography (PE/EA = 2/3) to give compound 105 (14.5 g, 19.87 mmol, 87.6% yield). ESI-MS: m/z 730.3 [M+H] ⁺

Synthesis of compound 106:

Compound 105 (14.5 g, 19.87 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and THF (150 mL) was added to the reaction mixture. The mixture was stirred until completely dissolved. A 5.4 mol/L sodium methoxide solution (5.5 mL, 29.82 mmol, 1.5 eq.) was added to the reaction mixture, and the mixture was stirred at room temperature for 3 hours until compound 105 was completely reacted. After the reaction was complete, the reaction mixture was concentrated under reduced pressure at 40 °C to obtain crude compound 106. The crude product was subjected to column chromatography to obtain compound 106 (12.0 g, 17.45 mmol, 87.8% yield). ESI-MS: m/z 688.3 [M+H] ⁺

Synthesis of compound 107:

The dried compound 106 (3.0 g, 4.36 mmol, 1.0 eq.) was added to a 50 mL round-bottom flask, and ultra-dry dichloromethane (30 mL) was added and stirred until completely dissolved. DIPEA (1.13 g, 8.72 mmol, 2.0 eq.) and DMAP (106.5 mg, 0.872 mmol, 0.2 eq.) were added to the reaction mixture, and the mixture was stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under nitrogen protection. CEP-Cl (1.55 g, 6.56 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature, and the reaction was carried out at room temperature for 30–60 minutes until compound 106 was completely reacted. The reaction mixture was quenched by adding a saturated aqueous solution of sodium bicarbonate, and the reaction mixture was extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude compound 107. The crude product was purified by column chromatography to give compound 107 (3.4 g, 3.83 mmol, 87.8% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.21(s,1H),8.75–8.54(m,2H),8.05(d,J＝7.3Hz,2H),7.68–7.50( m,3H),7.39–7.31(m,2H),7.29–7.15(m,7H),6.86–6.72(m,4H),6.05(d d,J＝34.0,3.2Hz,1H),4.74–4.30(m,2H),3.87–3.78(m,1H),3.79–3.4 6(m,12H),3.30(d,J＝24.0,3H),2.70–2.57(m,2H),1.11–0.87(m,12H), ^31P NMR (162MHz, DMSO-d ₆ )δ150.68,149.45.ESI-MS: m/z 888.4[M+H] ⁺

1.16: Synthesis of Compound 112

The following process describes the synthesis process of compound 112. Compound 112 is synthesized according to the process.

Synthesis of compound 108:

Compound 92 (15.0 g, 0.04 mol, 1.00 eq.) was added to a 500 mL three-necked round-bottom flask, and ultra-dry acetonitrile (150 mL) was added and stirred to dissolve. Then, BSA (24.4 g, 0.12 mol, 3.0 eq.) and GiBu (17.70 g, 0.08 mol, 2.0 eq.) were added. The reaction system was placed in an oil bath and heated to 80 °C, and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen protection. After the reaction was complete, the reaction system was placed in an ice-water bath at 0 °C and stirred for 30 minutes. TMSOTf (8.9 g, 0.04 mol, 1.0 eq.) was slowly added dropwise to the reaction. After the addition was complete, the reaction was placed in an oil bath and slowly heated to 80 °C, and reacted overnight at this temperature. TLC and LCMS analysis showed that compound 92 reacted completely. The reaction mixture was removed from the oil bath and cooled to room temperature. The reaction was quenched by adding a saturated sodium bicarbonate aqueous solution. The system was extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give crude compound 108. The crude product was subjected to column chromatography (PE/EA = 1/1) to give compound 108 (10.62 g, 21.26 mmol, 48.0% yield). ESI-MS: m/z 500.2 [M+H] ⁺

Synthesis of compound 109:

Compound 108 (10.62 g, 21.26 mmol, 1.0 eq.) was added to a 500 mL three-necked flask, and ultra-dry dichloromethane (100 mL) was added to the reaction flask. The reaction system was cooled to -10 °C and stirred at this temperature for 30 min. A 1.0 mol/L boron trichloride solution in dichloromethane (31.89 mL, 31.92 mmol, 1.5 eq.) was slowly added dropwise to the reaction system, and the reaction was continued for 5 h until compound 108 was completely reacted. The reaction was quenched at -10 °C with triethylamine and methanol. The reaction was brought back to room temperature, and water was added. The mixture was extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude compound 109. Crude compound 109 was subjected to column chromatography (PE/EA = 1/2) to obtain compound 109 (2.03 g, 4.96 mmol, 23.3% yield). ESI-MS: m/z 410.2 [M+H] ⁺

Synthesis of compound 110:

Compound 109 (2.0 g, 4.89 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DCE (25 mL) was added to the flask. The mixture was stirred until completely dissolved. DMTrCl (3.32 g, 9.78 mmol, 2.0 eq.), silver nitrate (0.83 g, 4.89 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (5.93 g, 48.9 mmol, 10.0 eq.) were added to the reaction mixture at room temperature and stirred until homogeneous. The reaction mixture was heated to 80 °C in an oil bath and allowed to react overnight. The reaction of compound 109 was confirmed to be complete by TLC and LCMS. The reaction mixture was then brought back to room temperature, and methanol was added to quench the reaction. The mixture was diluted with ethyl acetate and filtered through diatomaceous earth to obtain a filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain crude compound 110. The crude product was subjected to column chromatography (PE/EA = 2/3) to give compound 110 (2.96 g, 4.16 mmol, 85.1% yield). ESI-MS: m/z 712.4 [M+H] ⁺

Synthesis of compound 111:

Compound 110 (2.96 g, 4.16 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and THF (30 mL) was added to the reaction mixture. The mixture was stirred until completely dissolved. A 5.4 mol/L sodium methoxide solution (2.31 mL, 12.48 mmol, 3.0 eq.) was added to the reaction mixture, and the mixture was stirred at room temperature for 3 hours until compound 110 was completely reacted. After the reaction was complete, the reaction mixture was concentrated under reduced pressure at 40 °C to obtain crude compound 111. The crude product was subjected to column chromatography to obtain compound 111 (2.54 g, 3.79 mmol, 91.1% yield). ESI-MS: m/z 670.4 [M+H] ⁺

Synthesis of compound 112:

Dry compound 111 (2.54 g, 3.79 mmol, 1.0 eq.) was added to a 50 mL round-bottom flask, and ultra-dry dichloromethane (30 mL) was added and stirred until completely dissolved. DIPEA (0.98 g, 7.58 mmol, 2.0 eq.) and DMAP (93 mg, 0.758 mmol, 0.2 eq.) were added to the reaction mixture, and the mixture was stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under nitrogen protection. Compound CEP-Cl (1.35 g, 5.69 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature, and the reaction was carried out at room temperature for 30–60 minutes until compound 111 was completely reacted. The reaction mixture was quenched by adding saturated aqueous sodium bicarbonate solution, and the reaction mixture was extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude compound 112. The crude product was purified by column chromatography to give compound 112 (2.57 g, 2.96 mmol, 77.8% yield). ^¹H NMR (400 MHz, DMSO- _d⁶ ) δ 12.708 (s, 1H), 11.59 (s, 1H), 8.21 (d, J = 29.6 Hz, 1H), 7.55–7.18 (m, 9H), 6.88–6.79 (m, 4H), 5.72 (dd, J = 38.6, 4.8 Hz, 1H), 5.25–5.12 (m, 1H), 4.50 (t, J = 5.5 Hz, 1H), 3.8 0–3.68(m,8H),3.62–3.35(m,3H),3.30(d,J＝24.0,3H),3.25–3.20(m,1H),3.12(d,J ＝8.4Hz,1H),2.78–2.65(m,1H),2.57(m,1H),2.44–2.35(m,1H),1.11–0.87(m,18H), ³¹ P NMR (162MHz, DMSO-d ₆ )δ151.65,148.95.ESI-MS: m/z 870.5[M+H] ⁺

1.17: Synthesis of Compound 125

The following process describes the synthesis of compound 125. Compound 125 was synthesized according to the process.

Synthesis of compound 115:

Compound 113 (20.0 g, 133.22 mmol, 1.0 eq.) was dissolved in DMF (200 mL). Imidazole (22.67 g, 333.06 mmol, 2.5 eq.) was added to the reaction system, and the system was cooled to 0 °C and stirred continuously for 30 minutes. Then, tert-butyldiphenylchlorosilane (40.28 g, 146.54 mmol, 1.1 eq.) was slowly added dropwise to the reaction system. After the addition was complete, the ice bath was removed, and the reaction was heated to 60 °C and allowed to proceed overnight. TLC analysis showed that compound 113 reacted completely. Water was added to the reaction system, followed by extraction twice with ethyl acetate. The organic phases were combined and washed with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 35.38 g of crude intermediate compound 114. Crude compound 114 was dissolved in acetone (300 mL), and p-toluenesulfonic acid (1.15 g, 6.66 mmol, 0.05 eq.) was added, followed by 2,2-dimethoxypropane (69.37 g, 666.1 mmol, 5.0 eq.). After the addition was complete, the reaction was stirred at room temperature. TLC analysis showed that the intermediate reaction was complete. Sodium bicarbonate solid was added to quench the p-toluenesulfonic acid, the solid was removed by filtration, and the mixture was concentrated to obtain 38.24 g of crude compound 115. The crude compound was subjected to column chromatography (PE/EA = 100/15) to obtain compound 115 (19.27 g, 44.96 mmol, 33.75% yield). ESI-MS: m/z 428.6 [M+H] ⁺

Synthesis of compound 116:

Compound 115 (19.27 g, 44.96 mmol, 1.0 eq.) was dissolved in DMF (200 mL), stirred until completely dissolved, and the reaction system was cooled to approximately 0 °C. 60% NaH (2.70 g, 67.44 mmol, 1.5 eq.) was slowly added dropwise to the reaction system, maintaining the temperature at approximately 0 °C. After the addition was complete, the reaction system was brought to room temperature and reacted overnight at room temperature. TLC analysis showed that the reaction was complete, and compound 115 reacted completely. The reaction system was slowly poured into a saturated ammonium chloride aqueous solution at approximately 0 °C. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude compound 116 (38.2 g), which was directly used in the next reaction step.

Synthesis of compound 117:

Crude compound 116 (38.2 g) was dissolved in THF (200 mL) and stirred until completely dissolved. TBAF (1 M in THF, 50 mL, 50 mmol, 1.11 eq.) was added to the reaction mixture, and the mixture was stirred overnight at room temperature until compound 116 reacted completely. The reaction solution was concentrated under reduced pressure to remove the solvent. The crude product was subjected to column chromatography (PE/EA = 10/3) to give compound 117 (10.77 g, 38.41 mmol, two-step yield 85.43%). ESI-MS: m/z 280.3 [M+H] ⁺

Synthesis of compound 118:

Compound 117 (45.00 g, 160.53 mmol, 1.00 eq.) was added to a 500 mL round-bottom flask, and acetonitrile (400 mL) was added to the flask and stirred to dissolve. Then, imidazole (38.25 g, 561.86 mmol, 3.5 eq.) and triphenylphosphine (50.53 g, 192.64 mmol, 1.2 eq.) were added, followed by the slow addition of iodine (48.9 g, 192.64 mmol, 1.2 eq.). After the addition was complete, the reaction system was placed in an oil bath and heated to 80 °C. The mixture was stirred for 3 hours. TLC and LCMS analysis showed that compound 117 had reacted completely. The reaction mixture was removed from the oil bath and cooled to room temperature. The reaction was quenched by adding an aqueous sodium sulfite solution. The mixture was extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude compound 118. The crude product was subjected to column chromatography (PE/EA = 1/4) to give compound 118 (54.40 g, 139.41 mmol, 86.8% yield). ESI-MS: m/z 390.3 [M+H] ⁺

Synthesis of compound 119:

Compound 118 (54.40 g, 139.41 mmol, 1.0 eq.) was dissolved in MeOH (500 mL). Potassium carbonate (19.34 g, 139.42 mmol, 1.0 eq.) and palladium on carbon (10% W, 5.4 g) were then added to the reaction solution, and the mixture was stirred at room temperature until compound 118 was completely reacted. The system was filtered through diatomaceous earth, concentrated, extracted twice with ethyl acetate, filtered to remove salt, and concentrated under reduced pressure to obtain crude compound 119. Crude compound 119 was subjected to column chromatography (PE/EA = 4/1) to obtain compound 119 (33.20 g, 125.61 mmol, 90.1% yield). ESI-MS: m/z 264.3 [M+H] ⁺

Synthesis of compound 120:

Compound 119 (33.20 g, 125.61 mmol, 1.0 eq) was added to a 500 mL round-bottom three-necked flask, and acetic acid (300 mL) and acetic anhydride (64.12 g, 628.05 mmol, 5.0 eq) were added to the reaction system, and the mixture was stirred until completely dissolved. The reaction mixture was cooled to 0 °C and stirred at this temperature for 30 minutes. Concentrated sulfuric acid (10 mL) was slowly added dropwise to the reaction system, maintaining the temperature between 0 and 5 °C. After the addition was complete, the reaction system was allowed to return to room temperature and stirred for 8 hours until compound 119 reacted completely. The reaction system was cooled to approximately -5 °C and neutralized with ammonia to a pH of approximately 7. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude compound 120. The crude product was subjected to column chromatography (PE/EA = 4/1) to give compound 120 (21.20 g, 68.76 mmol, 54.7% yield). ESI-MS: m/z 308.3 [M+H] ⁺

Synthesis of compound 121:

Compound 120 (10.00 g, 32.43 mmol, 1.00 eq.) and N6-benzoyladenine (15.52 g, 64.86 mmol, 2.0 eq.) were added to a 500 mL three-necked round-bottom flask, and ultra-dry acetonitrile (150 mL) was added and stirred to dissolve. Then, BSA (19.79 g, 97.29 mmol, 3.0 eq.) was added. The reaction mixture was heated to 80 °C in an oil bath and stirred at this temperature for 1 hour under nitrogen protection. After the reaction was complete, the mixture was placed in an ice-water bath at 0 °C and stirred for 30 minutes. TMSOTf (7.21 g, 32.43 mmol, 1.0 eq.) was slowly added dropwise to the reaction mixture. After the addition was complete, the mixture was placed in an oil bath and slowly heated to 80 °C, and reacted overnight at this temperature. TLC and LCMS analysis showed that compound 120 reacted completely. The reaction mixture was removed from the oil bath and cooled to room temperature. The reaction was quenched by adding a saturated sodium bicarbonate aqueous solution, and the mixture was extracted with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give crude compound 121. The crude product was subjected to column chromatography (PE/EA = 1/5) to give compound 121 (11.40 g, 23.38 mmol, 72.1% yield). ESI-MS: m/z 487.5 [M+H] ⁺

Synthesis of compound 122:

Compound 121 (11.40 g, 23.38 mmol, 1.0 eq.) was added to a 500 mL three-necked flask, and ultra-dry dichloromethane (100 mL) was added to the reaction flask. The reaction system was cooled to -78 °C and stirred at this temperature for 30 minutes. Boron trichloride (1 M in toluene, 70 mL, 70.14 mmol, 3.0 eq.) was slowly added dropwise to the reaction system. After the addition was complete, the temperature was restored to -10 °C and the reaction continued until compound 121 was completely reacted. The reaction was quenched at -78 °C with triethylamine and methanol. The reaction was brought back to room temperature and water was added. The mixture was extracted with dichloromethane, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum evaporation to obtain crude compound 122. The crude compound 122 was subjected to column chromatography (PE/EA = 1/10) to give compound 122 (4.80 g, 12.08 mmol, 51.67% yield). ESI-MS: m/z 397.3 [M+H] ⁺

Synthesis of compound 123:

Compound 122 (4.80 g, 12.08 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask, and ultra-dry DCE (50 mL) was added to the flask. The mixture was stirred until completely dissolved. DMTrCl (8.19 g, 24.16 mmol, 2.0 eq.), silver nitrate (2.05 g, 12.08 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (7.32 g, 60.40 mmol, 5.0 eq.) were added to the reaction mixture at room temperature and stirred until homogeneous. The reaction mixture was heated to 80 °C in an oil bath and allowed to react overnight. The reaction of compound 122 was confirmed to be complete by TLC and LCMS. The reaction mixture was then brought back to room temperature, and methanol was added to quench the reaction. The mixture was diluted with ethyl acetate and filtered through diatomaceous earth to obtain a filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain crude compound 123. The crude product was subjected to column chromatography (PE/EA = 1/3) to give compound 123 (6.20 g, 8.86 mmol, 73.34% yield). ESI-MS: m/z 699.8 [M+H] ⁺

Synthesis of compound 124:

Compound 123 (6.20 g, 8.86 mmol, 1.0 eq.) was dissolved in THF (100 mL), followed by the addition of sodium methoxide (574 mg, 10.63 mmol, 1.2 eq.). The mixture was stirred at room temperature until compound 123 was completely reacted. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude compound 124. Crude compound 124 was subjected to column chromatography (PE/EA = 1/5) to obtain compound 124 (5.21 g, 7.91 mmol, 90% yield). ESI-MS: m/z 657.7 [M+H] ⁺

Synthesis of compound 125:

The dried compound 124 (5.21 g, 7.91 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry dichloromethane (50 mL) was added and stirred until completely dissolved. DIPEA (2.04 g, 15.82 mmol, 2.0 eq.) and DMAP (193 mg, 1.58 mmol, 0.2 eq.) were added to the reaction mixture, and the mixture was stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under nitrogen protection. CEP-Cl (2.81 g, 11.87 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature, and the mixture was reacted at room temperature for 30–60 minutes until compound 124 was completely reacted. The reaction system was quenched by adding a saturated aqueous solution of sodium bicarbonate, and the reaction mixture was extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude compound 125. The crude product was purified by column chromatography (PE/EA = 1/3) to give compound 125 (5.0 g, 5.83 mmol, 74% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ10.17(s,1H),8.40(s,1H),8.15(s,1H),8.03–7.86(m,2H),7.56–7. 41(m,3H),7.14–7.32(m,9H),6.78–6.72(m,4H),6.35(t,J＝9.0Hz,1H) ,5.01(s,1H),4.48–4.28(m,1H),3.79–3.65(m,3H),3.72(s,6H),2.96 –2.74(m,2H),2.73–2.66(m,2H),1.13(s,3H),1.06(d,J＝12.0Hz,12H). ^31P NMR(162MHz, DMSO-d ₆ )δ152.81(s),151.7(s).ESI-MS: m/z 858.3[M+H] ⁺

1.18: Synthesis of Compound 129

The following process describes the synthesis of compound 129. Compound 129 is synthesized according to the process (during the synthesis of the nucleic acid sequence, the 5' Ac group is removed to obtain -OH).

Synthesis of compound 20:

Compound 4 (8.0 g, 21.84 mmol, 1.0 eq.) and N6-benzoyladenine (10.45 g, 43.68 mmol, 2.0 eq.) were added to a 500 mL three-necked round-bottom flask, and ultra-dry acetonitrile (150 mL) was added and stirred to dissolve. Then, BSA (13.33 g, 65.52 mmol, 3.0 eq.) was added. The reaction mixture was heated to 80 °C in an oil bath and stirred at this temperature for 1 hour under nitrogen protection. After the reaction was complete, the mixture was placed in an ice-water bath at 0 °C and stirred for 30 minutes. TMSOTf (4.85 g, 21.84 mmol, 1.0 eq.) was slowly added dropwise to the reaction mixture. After the addition was complete, the mixture was placed in an oil bath and slowly heated to 80 °C, and reacted overnight at this temperature. TLC and LCMS analysis showed that compound 4 reacted completely. The reaction mixture was removed from the oil bath and cooled to room temperature. The reaction was quenched by adding a saturated aqueous sodium bicarbonate solution, and the mixture was extracted with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give crude compound 20. The crude product was subjected to column chromatography (PE/EA = 1/5) to give compound 20 (8.50 g, 15.58 mmol, 71.34% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.17(s,1H),8.35(s,1H),8.21(s,1H),7.99–7.89(m,2H),7.67–7.47(m,3H),7.39–7.21(m,5H),6.13(d,J＝1 4.8Hz,1H),5.43(t,J＝15.3Hz,1H),4.61–4.41(m,4H),4.15–3.80(m,2H),2.01(s,3H),1.99(s,3H).ESI-MS:m/z 546.2[M+H] ⁺

Synthesis of compound 126:

Compound 20 (8.50 g, 15.58 mmol, 1.0 eq.) was added to a 500 mL three-necked flask, and ultra-dry dichloromethane (100 mL) was added to the reaction flask. The reaction system was cooled to -78 °C and stirred at this temperature for 30 minutes. Boron trichloride (1 M in toluene, 46.74 mL, 46.74 mmol, 3.0 eq.) was slowly added dropwise to the reaction system. After the addition was complete, the temperature was restored to -10 °C and the reaction continued until complete. The reaction was quenched at -78 °C with triethylamine and methanol. The reaction was brought back to room temperature and water was added. The mixture was extracted with dichloromethane, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude compound 126. Crude compound 126 was subjected to column chromatography (PE/EA = 1/10) to obtain compound 126 (4.80 g, 10.54 mmol, 67.65% yield). ESI-MS: m/z 456.2 [M+H] ⁺

Synthesis of compound 127:

Compound 126 (4.80 g, 10.54 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask, and ultra-dry DCE (80 mL) was added to the flask. The mixture was stirred until completely dissolved. DMTrCl (7.14 g, 21.08 mmol, 2.0 eq.), silver nitrate (1.79 g, 10.54 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (6.39 g, 52.70 mmol, 5.0 eq.) were added to the reaction mixture at room temperature and stirred until homogeneous. The reaction mixture was heated to 80 °C in an oil bath and allowed to react overnight. The reaction of compound 126 was confirmed to be complete by TLC and LCMS. The reaction mixture was brought back to room temperature, and methanol was added to quench the reaction. The mixture was diluted with ethyl acetate and filtered through diatomaceous earth to obtain a filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain crude compound 127. The crude product was subjected to column chromatography (PE/EA = 1/5) to give compound 127 (6.33 g, 8.35 mmol, 79.22% yield). ESI-MS: m/z 756.3 [MH ^]

Synthesis of compound 128:

Compound 127 (6.33 g, 8.35 mmol, 1.0 eq.) was dissolved in THF (70 mL). The reaction solution was cooled in an ice bath, followed by the addition of 10 mL of ammonia. The reaction was maintained at 0 °C and stirred until complete. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give crude compound 128. Crude compound 128 was subjected to column chromatography (PE/EA = 1/8) to give compound 128 (2.58 g, 3.60 mmol, 43.11% yield). ^¹H NMR (400 MHz, DMSO- _d6) was used. )δ11.17(s,1H),8.35(s,1H),8.21(s,1H),8.01–7.87(m,2H),7.69–7.47(m,3H),7 .33–7.22(m,9H),6.94–6.82(m,4H),5.90(t,J＝9.0Hz,1H),5.23(d,J＝3.8Hz,1H), 5.18(s,1H),4.42(dd,J＝24.8,14.5Hz,1H),4.31–4.18(m,1H),4.04(dd,J＝24.8,1 4.5Hz,1H),3.94(d,J＝3.4Hz,1H),3.82(s,6H),2.03(s,1H).ESI-MS:m/z714.3[MH] ^-

Synthesis of compound 129:

The dried compound 128 (2.58 g, 3.60 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry dichloromethane (30 mL) was added and stirred until completely dissolved. DIPEA (930 mg, 7.20 mmol, 2.0 eq.) and DMAP (88 mg, 0.72 mmol, 0.2 eq.) were added to the reaction mixture, and the mixture was stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under nitrogen protection. CEP-Cl (1.28 g, 5.4 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature, and the mixture was reacted at room temperature for 30–60 minutes until compound 128 was completely reacted. The reaction was quenched by adding a saturated aqueous solution of sodium bicarbonate, and the reaction mixture was extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude compound 129. The crude product was purified by column chromatography (PE/EA = 1/3) to give compound 129 (2.73 g, 2.98 mmol, 82.78% yield). ^¹H NMR (400 MHz, DMSO- _d₆ ) δ 11.17 (s, 1H), 8.35 (s, 1H), 8.21 (s, 1H), 8.01–7.87 (m, 2H), 7.69–7.47 (m, 3H), 7.33–7.22 (m, 9H), 6.94–6.82 (m, 4H), 5.90 (t, J = 9.0 Hz, 1H), 5.18 (s, 1H), 4.42 (dd, J = 24.8 Hz). ,14.5Hz,1H),4.31–4.18(m,1H),4.04(dd,J＝24.8,14.5Hz,1H),3.99–3.85(m,3H),3. 82(s,6H),2.96–2.74(m,2H),2.73–2.66(m,2H),2.03(s,3H),1.06(d,J＝12.0Hz,12H). ³¹ P NMR (162MHz, DMSO-d ₆ )δ150.54(s),149.35(s).ESI-MS: m/z 914.4[MH] ^-

1.19: Synthesis of Compound 136

The following process describes the synthesis of compound 136. Compound 136 was synthesized according to the process.

Synthesis of compound 130:

Compound 42 (15.97 g, 56.97 mmol, 1.0 eq.) was dissolved in DCM (160 mL) and stirred until completely dissolved. The solution was then cooled in a -78 °C bath. Pyridine (22.53 g, 284.85 mmol, 5.0 eq.) was added to the reaction mixture, followed by the slow dropwise addition of diethylaminotrifluoride (18.37 g, 113.94 mmol, 2.0 eq.). After the addition was complete, the mixture was slowly heated to room temperature and stirred overnight at room temperature until compound 42 reacted completely. The reaction mixture was quenched with methanol, and an aqueous sodium bicarbonate solution was added. The mixture was then extracted twice with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude compound 130. The crude product was subjected to column chromatography (PE/EA = 5/1) to obtain compound 130 (11.38 g, 40.31 mmol, 70.76% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ7.42–7.26(m,5H),5.85(d,J＝3.6Hz,1H),4.69(m,2H),4.52–4.41(d,J＝12Hz,1H ),4.03(m,1H),3.81(d,J＝3.2Hz,1H),3.70–3.51(m,2H),1.32(s,3H),1.25(s,3H). ¹⁹ F NMR (377MHz, DMSO-d ₆ ): δ-229.73.ESI-MS: m/z 281.1[M+H] ⁺

Synthesis of compound 131:

Compound 130 (11.38 g, 40.31 mmol, 1.0 eq.) was added to a 500 mL round-bottom three-necked flask, and acetic acid (190 mL) and acetic anhydride (20.58 g, 201.55 mmol, 5.0 eq.) were added to the reaction system, and the mixture was stirred until completely dissolved. The reaction mixture was cooled to 0 °C and stirred at this temperature for 30 minutes. Concentrated sulfuric acid (2.0 mL) was slowly added dropwise to the reaction system, maintaining the temperature between 0 and 5 °C. After the addition was complete, the reaction system was allowed to return to room temperature and stirred for 8 hours until compound 130 reacted completely. The reaction system was cooled to approximately -5 °C and neutralized with ammonia to a pH of approximately 7. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude compound 131. The crude compound 131 was subjected to column chromatography (PE/EA = 4/1) to give compound 131 (6.32 g, 19.37 mmol, 48.05% yield). ESI-MS: m/z 327.1 [M+H] ⁺

Synthesis of compound 132:

Compound 131 (6.32 g, 19.37 mmol, 1.0 eq.) and N6-benzoyladenine (9.27 g, 38.74 mmol, 2.0 eq.) were added to a 500 mL three-necked round-bottom flask, and ultra-dry acetonitrile (150 mL) was added and stirred to dissolve. Then, BSA (11.82 g, 58.11 mmol, 3.0 eq.) was added. The reaction system was heated to 80 °C in an oil bath and stirred at this temperature for 1 hour under nitrogen protection. After the reaction was complete, the reaction system was placed in an ice-water bath at 0 °C and stirred for 30 minutes. TMSOTf (4.31 g, 19.37 mmol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition was complete, the reaction system was placed in an oil bath and slowly heated to 80 °C, and reacted overnight at this temperature. TLC and LCMS analysis showed that compound 131 reacted completely. The reaction mixture was removed from the oil bath and cooled to room temperature. A saturated aqueous sodium bicarbonate solution was added to quench the reaction, and the mixture was extracted with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give crude compound 132. The crude product was subjected to column chromatography (PE/EA = 1/5) to give compound 132 (6.50 g, 12.86 mmol, 66.39% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.17(s,1H),8.35(s,1H),8.21(s,1H),7.99–7.89(m,2H),7.67–7.47(m,3H),7.39–7.21(m,5H) ,6.13(d,J＝14.8Hz,1H),5.43(t,J＝15.3Hz,1H),4.61–4.41(m,4H),4.15–3.80(m,2H),1.99(s,3H). ¹⁹ F NMR (377MHz, DMSO-d ₆ ): δ-229.71.ESI-MS: m/z 506.2[M+H] ⁺

Synthesis of compound 133:

Compound 132 (6.50 g, 12.88 mmol, 1.0 eq.) was added to a 250 mL three-necked flask, and ultra-dry dichloromethane (70 mL) was added to the reaction flask. The reaction system was cooled to -78 °C and stirred at this temperature for 30 minutes. Boron trichloride (1 M in toluene, 38.64 mL, 38.64 mmol, 3.0 eq.) was slowly added dropwise to the reaction system. After the addition was complete, the temperature was restored to -10 °C and the reaction continued until compound 132 was completely reacted. The reaction was quenched at -78 °C with triethylamine and methanol. The reaction was brought back to room temperature and water was added. The mixture was extracted with dichloromethane, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude compound 133. The crude compound 133 was subjected to column chromatography (PE/EA = 1/8) to give compound 133 (3.12 g, 7.51 mmol, 58.31% yield). ESI-MS: m/z 416.1 [M+H] ⁺

Synthesis of compound 134:

Compound 133 (3.12 g, 7.51 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DCE (50 mL) was added to the flask. The mixture was stirred until completely dissolved. DMTrCl (5.09 g, 15.02 mmol, 2.0 eq.), silver nitrate (1.28 g, 7.51 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (4.55 g, 37.55 mmol, 5.0 eq.) were added to the reaction mixture at room temperature and stirred until homogeneous. The reaction mixture was heated to 80 °C in an oil bath and allowed to react overnight. The reaction of compound 133 was confirmed to be complete by TLC and LCMS. The reaction mixture was then brought back to room temperature, and methanol was added to quench the reaction. The mixture was diluted with ethyl acetate and filtered through diatomaceous earth to obtain a filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain crude compound 134. The crude product was subjected to column chromatography (PE/EA = 1/5) to give compound 134 (4.63 g, 6.45 mmol, 85.89% yield). ESI-MS: m/z 716.3 [MH ^]

Synthesis of compound 135:

Compound 134 (4.63 g, 6.45 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and THF (50 mL) was added to the reaction mixture. The mixture was stirred until completely dissolved. A 5.4 mol/L sodium methoxide solution (1.43 mL, 7.74 mmol, 1.2 eq.) was added to the reaction mixture, and the mixture was stirred at room temperature until compound 134 was completely reacted. After the reaction was complete, the reactants were extracted twice with water and ethyl acetate. The combined organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude compound 135. The crude compound was subjected to column chromatography to obtain compound 135 (4.14 g, 6.13 mmol, 95.04% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.17(s,1H),8.35(s,1H),8.21(s,1H),8.01–7.87(m,2H),7.69–7.47(m,3H),7.33–7.22(m,9H),6.94–6.82(m,4 H), 5.90 (t, J = 9.0Hz, 1H), 5.23 (d, J = 3.8Hz, 1H), 5.18 (s, 1H), 4.42–4.04 (m, 3H), 3.94 (d, J = 3.4Hz, 1H), 3.82 (s, 6H). ¹⁹ F NMR (377MHz, DMSO-d ₆ ): δ-229.64.ESI-MS: m/z 674.2[MH] ^-

Synthesis of compound 136:

The dried compound 135 (4.14 g, 6.13 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry dichloromethane (50 mL) was added and stirred until completely dissolved. DIPEA (1.58 g, 12.26 mmol, 2.0 eq.) and DMAP (150 mg, 1.23 mmol, 0.2 eq.) were added to the reaction mixture, and the mixture was stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under nitrogen protection. CEP-Cl (2.18 g, 9.20 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature, and the reaction was continued at room temperature for 30–60 minutes until compound 135 was completely reacted. The reaction mixture was quenched by adding a saturated aqueous solution of sodium bicarbonate, and the reaction mixture was extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude compound 136. The crude product was purified by column chromatography (PE/EA = 1/3) to give compound 136 (4.54 g, 5.18 mmol, 84.50% yield). ^¹H NMR (400 MHz, DMSO- _d₆ ) δ 11.17 (s, 1H), 8.35 (s, 1H), 8.21 (s, 1H), 8.01–7.87 (m, 2H), 7.69–7.47 (m, 3H), 7.33–7.22 (m, 9H), 6.94–6.82 (m, 4H), 5.90 (t, J = 9.0 Hz, 1H), 5.18 (s, 1H), 4.42 (dd, J) ＝24.8,14.5Hz,1H),4.31–4.18(m,1H),4.04(dd,J＝24.8,14.5Hz,1H),3.99–3.85( m,3H),3.82(s,6H),2.96–2.74(m,3H),2.73–2.66(m,3H),1.06(d,J＝12.0Hz,12H). ¹⁹ F NMR (377MHz, DMSO-d ₆ ): δ-229.69. ³¹ P NMR (162MHz, DMSO-d ₆ ) δ 150.71 (s), 149.62 (s). ESI-MS: m/z 874.4 [MH] ^-

1.20: Synthesis of Compound 142

The following process describes the synthesis of compound 142. Compound 142 is synthesized according to the process (when synthesizing the nucleic acid sequence, the Ac group at 5' is removed to obtain -OH).

Synthesis of compound 137:

Crude compound 116 (33.38 g) was added to a 500 mL round-bottom three-necked flask, and acetic acid (260 mL) and acetic anhydride (27.54 g, 269.76 mmol, 6.0 eq.) were added to the reaction system, and the mixture was stirred until completely dissolved. The reaction mixture was cooled to 0 °C and stirred at this temperature for 30 minutes. Concentrated sulfuric acid (5 mL) was slowly added dropwise to the reaction system, maintaining the temperature between 0 and 5 °C. After the addition was complete, the reaction system was allowed to return to room temperature and stirred for 8 hours until the reaction was complete. The reaction system was cooled to approximately -5 °C and neutralized with ammonia to a pH of approximately 7. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude compound 137. The crude product was subjected to column chromatography (PE/EA = 4/1) to give compound 137 (10.55 g, 28.80 mmol, overall two-step yield: 64.06%). ^¹H NMR (400 MHz, DMSO- _d⁶ ) δ 7.36–7.26 (m, 20H), 6.08–6.02 (m, 4H), 5.51–5.42 (m, 4H), 4.84–4.63 (m, 1H), 4.59–4.48 (m, 1H), 4.31–4.12 (m, 1H), 4.01–3.81 (m, 1H), 2.05 (s, 12H), 2.15–1.96 (m, 2H). ESI-MS: m/z 367.1 [M+H] ^⁺

Synthesis of compound 138:

Compound 137 (8.0 g, 21.84 mmol, 1.0 eq.) and N6-benzoyladenine (10.45 g, 43.68 mmol, 2.0 eq.) were added to a 500 mL three-necked round-bottom flask, and ultra-dry acetonitrile (150 mL) was added and stirred to dissolve. Then, BSA (13.33 g, 65.52 mmol, 3.0 eq.) was added. The reaction mixture was heated to 80 °C in an oil bath and stirred at this temperature for 1 hour under nitrogen protection. After the reaction was complete, the mixture was placed in an ice-water bath at 0 °C and stirred for 30 minutes. TMSOTf (4.85 g, 21.84 mmol, 1.0 eq.) was slowly added dropwise to the reaction mixture. After the addition was complete, the mixture was placed in an oil bath and slowly heated to 80 °C, and reacted overnight at this temperature. TLC and LCMS analysis showed that compound 137 reacted completely. The reaction mixture was removed from the oil bath and cooled to room temperature. The reaction was quenched by adding a saturated aqueous sodium bicarbonate solution, and the mixture was extracted with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give crude compound 138. The crude product was subjected to column chromatography (PE/EA = 1/5) to give compound 138 (8.85 g, 16.22 mmol, 74.27% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.18(s,1H),8.36(s,1H),8.22(s,1H),7.98–7.87(m,2H),7.67–7.46(m,3H),7.37–7.20(m,5H),6.12(d,J＝1 4.8Hz,1H),5.41(t,J＝15.3Hz,1H),4.62–4.43(m,4H),4.14–3.81(m,2H),2.02(s,3H),1.98(s,3H).ESI-MS:m/z 546.2[M+H] ⁺

Synthesis of compound 139:

Compound 138 (8.85 g, 16.22 mmol, 1.0 eq.) was added to a 500 mL three-necked flask, and ultra-dry dichloromethane (100 mL) was added to the reaction flask. The reaction system was cooled to -78 °C and stirred at this temperature for 30 minutes. Boron trichloride (1 M in toluene, 48.66 mL, 48.66 mmol, 3.0 eq.) was slowly added dropwise to the reaction system. After the addition was complete, the temperature was restored to -10 °C and the reaction continued until compound 138 was completely reacted. The reaction was quenched at -78 °C with triethylamine and methanol. The reaction was brought back to room temperature and water was added. The mixture was extracted with dichloromethane, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude compound 139. The crude compound 139 was subjected to column chromatography (PE/EA = 1/10) to yield compound 139 (4.39 g, 9.64 mmol, 59.43% yield). ESI-MS: m/z 456.2 [M+H] ⁺

Synthesis of compound 140:

Compound 139 (4.39 g, 9.64 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DCE (50 mL) was added to the flask. The mixture was stirred until completely dissolved. DMTrCl (6.53 g, 19.28 mmol, 2.0 eq.), silver nitrate (1.64 g, 9.64 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (5.84 g, 48.20 mmol, 5.0 eq.) were added to the reaction mixture at room temperature and stirred until homogeneous. The reaction mixture was heated to 80 °C in an oil bath and allowed to react overnight. The reaction of compound 139 was confirmed to be complete by TLC and LCMS. The reaction mixture was brought back to room temperature, and methanol was added to quench the reaction. The mixture was diluted with ethyl acetate and filtered through diatomaceous earth to obtain a filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain crude compound 140. The crude product was subjected to column chromatography (PE/EA = 1/5) to give compound 140 (6.11 g, 8.06 mmol, 84.65% yield). ESI-MS: m/z 756.3 [MH ^]

Synthesis of compound 141:

Compound 140 (6.11 g, 8.06 mmol, 1.0 eq.) was dissolved in THF (60 mL). The reaction solution was cooled in an ice bath, followed by the addition of 8.6 mL of ammonia. The reaction was maintained at 0 °C and stirred until compound 140 was completely reacted. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give crude compound 141. Column chromatography (PE/EA = 1/8) yielded compound 141 (1.98 g, 2.77 mmol, 34.37% yield). ^¹H NMR (400 MHz, DMSO- _d6) was used. )δ11.19(s,1H),8.36(s,1H),8.22(s,1H),8.00–7.86(m,2H),7.68–7.48(m,3H),7 .31–7.23(m,9H),6.93–6.83(m,4H),5.91(t,J＝9.0Hz,1H),5.22(d,J＝3.8Hz,1H), 5.19(s,1H),4.43(dd,J＝24.8,14.5Hz,1H),4.32–4.19(m,1H),4.03(dd,J＝24.8,1 4.5Hz,1H),3.95(d,J＝3.4Hz,1H),3.83(s,6H),2.02(s,1H).ESI-MS:m/z714.3[MH] ^-

Synthesis of compound 142:

The dried compound 141 (1.98 g, 2.77 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry dichloromethane (20 mL) was added and stirred until completely dissolved. DIPEA (716 mg, 5.54 mmol, 2.0 eq.) and DMAP (67 mg, 0.55 mmol, 0.2 eq.) were added to the reaction mixture, and the mixture was stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under nitrogen protection. CEP-Cl (985 mg, 4.16 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature, and the reaction was carried out at room temperature for 30–60 minutes until complete. The reaction was quenched by adding a saturated aqueous solution of sodium bicarbonate, and the reaction mixture was extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude compound 142. The crude product was purified by column chromatography (PE/EA = 1/3) to give compound 142 (2.08 g, 2.27 mmol, 81.95% yield). ^¹H NMR (400 MHz, DMSO- _d₆ ) δ 11.18 (s, 1H), 8.36 (s, 1H), 8.22 (s, 1H), 8.00–7.88 (m, 2H), 7.68–7.48 (m, 3H), 7.34–7.21 (m, 9H), 6.95–6.81 (m, 4H), 5.91 (t, J = 9.0 Hz, 1H), 5.12 (s, 1H), 4.42 (dd, J = 24.8 Hz). ,14.5Hz,1H),4.32–4.19(m,1H),4.03(dd,J＝24.8,14.5Hz,1H),3.98–3.84(m,3H),3. 81(s,6H),2.96–2.73(m,2H),2.74–2.65(m,2H),2.02(s,3H),1.07(d,J＝12.0Hz,12H). ³¹ P NMR (162MHz, DMSO-d ₆ )δ150.31(s),149.22(s).ESI-MS: m/z914.4[MH] ^-

1.21: Synthesis of Compound 149

The following process describes the synthesis of compound 149. Compound 149 was synthesized according to the process.

Synthesis of compound 143:

Compound 117 (16.77 g, 59.82 mmol, 1.0 eq.) was dissolved in DCM (160 mL) and stirred until completely dissolved. The solution was then cooled in a -78 °C bath. Pyridine (23.66 g, 299.10 mmol, 5.0 eq.) was added to the reaction mixture, followed by the slow dropwise addition of diethylaminotrifluoride (19.28 g, 119.64 mmol, 2.0 eq.). After the addition was complete, the mixture was slowly heated to room temperature and stirred overnight at room temperature until compound 117 reacted completely. The reaction mixture was quenched with methanol, and an aqueous sodium bicarbonate solution was added. The mixture was then extracted twice with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude compound 143. The crude product was subjected to column chromatography (PE/EA = 5/1) to obtain compound 143 (12.33 g, 43.68 mmol, 73.02% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ7.41–7.25(m,5H),5.86(d,J＝3.6Hz,1H),4.71–4.62(m,2H),4.45(d,J＝12Hz,1H ),4.05(m,1H),3.82(d,J＝3.2Hz,1H),3.71–3.52(m,2H),1.33(s,3H),1.24(s,3H). ¹⁹ F NMR (377MHz, DMSO-d ₆ ): δ-229.66.ESI-MS: m/z 281.1[M+H] ⁺

Synthesis of compound 144:

Compound 143 (12.33 g, 43.68 mmol, 1.0 eq.) was added to a 500 mL round-bottom three-necked flask, and acetic acid (210 mL) and acetic anhydride (22.30 g, 218.40 mmol, 5.0 eq.) were added to the reaction system, and the mixture was stirred until completely dissolved. The reaction mixture was cooled to 0 °C and stirred at this temperature for 30 minutes. Concentrated sulfuric acid (2.5 mL) was slowly added dropwise to the reaction system, maintaining the temperature between 0 and 5 °C. After the addition was complete, the reaction system was allowed to return to room temperature and stirred for another 8 hours until compound 143 had completely reacted. The reaction system was cooled to approximately -5 °C and neutralized with ammonia to a pH of approximately 7. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude compound 144. The crude compound 144 was subjected to column chromatography (PE/EA = 4/1) to give compound 144 (6.98 g, 21.39 mmol, 48.97% yield). ESI-MS: m/z 327.1 [M+H] ⁺

Synthesis of compound 145:

Compound 144 (6.98 g, 21.39 mmol, 1.0 eq.) and N6-benzoyladenine (20.23 g, 42.78 mmol, 2.0 eq.) were added to a 500 mL three-necked round-bottom flask, and ultra-dry acetonitrile (200 mL) was added and stirred to dissolve. Then, BSA (13.05 g, 64.17 mmol, 3.0 eq.) was added. The reaction mixture was heated to 80 °C in an oil bath and stirred at this temperature for 1 hour under nitrogen protection. After the reaction was complete, the mixture was placed in an ice-water bath at 0 °C and stirred for 30 minutes. TMSOTf (4.75 g, 21.39 mmol, 1.0 eq.) was slowly added dropwise to the reaction mixture. After the addition was complete, the mixture was placed in an oil bath and slowly heated to 80 °C, and reacted overnight at this temperature. TLC and LCMS analysis showed that compound 144 reacted completely. The reaction mixture was removed from the oil bath and cooled to room temperature. A saturated aqueous sodium bicarbonate solution was added to quench the reaction, and the mixture was extracted with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give crude compound 145. The crude product was subjected to column chromatography (PE/EA = 1/5) to give compound 145 (7.12 g, 14.08 mmol, 65.83% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ11.18(s,1H),8.36(s,1H),8.22(s,1H),7.98–7.87(m,2H),7.66–7.46(m,3H),7.38–7.20(m,5H) ,6.12(d,J＝14.8Hz,1H),5.42(t,J＝15.3Hz,1H),4.62–4.40(m,4H),4.16–3.81(m,2H),1.99(s,3H). ¹⁹ F NMR (377MHz, DMSO-d ₆ ): δ-229.77.ESI-MS: m/z 506.2[M+H] ⁺

Synthesis of compound 146:

Compound 145 (7.12 g, 14.08 mmol, 1.0 eq.) was added to a 250 mL three-necked flask, and ultradry dichloromethane (70 mL) was added to the reaction flask. The reaction system was cooled to -78 °C and stirred at this temperature for 30 min. Boron trichloride (1 M in toluene, 42.24 mL, 42.24 mmol, 3.0 eq.) was slowly added dropwise to the reaction system. After the addition was complete, the temperature was restored to -10 °C and the reaction continued until complete. The reaction was quenched at -78 °C with triethylamine and methanol. The reaction was brought back to room temperature and water was added. The mixture was extracted with dichloromethane, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude compound 146. Crude compound 146 was subjected to column chromatography (PE/EA = 1/8) to obtain compound 146 (3.82 g, 9.20 mmol, 65.34% yield). ESI-MS: m/z 416.1 [M+H] ⁺

Synthesis of compound 147:

Compound 146 (3.82 g, 9.20 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DCE (50 mL) was added to the flask. The mixture was stirred until completely dissolved. DMTrCl (6.23 g, 18.40 mmol, 2.0 eq.), silver nitrate (1.56 g, 9.20 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (5.57 g, 46.00 mmol, 5.0 eq.) were added to the reaction mixture at room temperature and stirred until homogeneous. The reaction mixture was heated to 80 °C in an oil bath and allowed to react overnight. The reaction of compound 146 was confirmed to be complete by TLC and LCMS. The reaction mixture was brought back to room temperature, and methanol was added to quench the reaction. The mixture was diluted with ethyl acetate and filtered through diatomaceous earth to obtain a filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain crude compound 147. The crude product was subjected to column chromatography (PE/EA = 1/5) to give compound 147 (4.81 g, 6.70 mmol, 72.83% yield). ESI-MS: m/z 716.3 [MH ^]

Synthesis of compound 148:

Compound 147 (4.81 g, 6.70 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and THF (50 mL) was added to the reaction mixture. The mixture was stirred until completely dissolved. A 5.4 mol/L sodium methoxide solution (1.49 mL, 8.04 mmol, 1.2 eq.) was added to the reaction mixture, and the mixture was stirred at room temperature until compound 147 was completely reacted. After the reaction was complete, the reaction mixture was extracted twice with water and ethyl acetate. The combined organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude compound 148. The crude product was subjected to column chromatography to obtain compound 148 (4.32 g, 6.39 mmol, 95.37% yield). ¹ HNMR (400MHz, DMSO-d ₆ )δ11.18(s,1H),8.36(s,1H),8.22(s,1H),8.02–7.88(m,2H),7.68–7.46(m,3H),7.32–7.21(m,9H),6.95–6.81(m,4 H), 5.91 (t, J = 9.0Hz, 1H), 5.24 (d, J = 3.8Hz, 1H), 5.19 (s, 1H), 4.43–4.03 (m, 3H), 3.95 (d, J = 3.4Hz, 1H), 3.81 (s, 6H). ¹⁹ F NMR (377MHz, DMSO-d ₆ ): δ-229.75.ESI-MS: m/z 674.2[MH] ^-

Synthesis of compound 149:

The dried compound 148 (4.32 g, 6.39 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry dichloromethane (50 mL) was added and stirred until completely dissolved. DIPEA (1.65 g, 12.78 mmol, 2.0 eq.) and DMAP (156 mg, 1.28 mmol, 0.2 eq.) were added to the reaction mixture, and the mixture was stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under nitrogen protection. CEP-Cl (2.27 g, 9.59 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature, and the mixture was reacted at room temperature for 30–60 minutes until complete. The reaction was quenched by adding a saturated aqueous solution of sodium bicarbonate, and the reaction mixture was extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude compound 149. The crude product was purified by column chromatography (PE/EA = 1/3) to give compound 149 (4.65 g, 5.31 mmol, 83.10% yield). ^¹H NMR (400 MHz, DMSO- _d₆ ) δ 11.18 (s, 1H), 8.36 (s, 1H), 8.22 (s, 1H), 8.02–7.86 (m, 2H), 7.68–7.45 (m, 3H), 7.32–7.21 (m, 9H), 6.95–6.81 (m, 4H), 5.91 (t, J = 9.0 Hz, 1H), 5.17 (s, 1H), 4.43 (dd, J) ＝24.8,14.5Hz,1H),4.32–4.17(m,1H),4.03(dd,J＝24.8,14.5Hz,1H),3.99–3.84( m,3H),3.81(s,6H),2.97–2.73(m,3H),2.72–2.65(m,3H),1.05(d,J＝12.0Hz,12H). ¹⁹ F NMR (377MHz, DMSO-d ₆ ): δ-229.72. ³¹ P NMR (162MHz, DMSO-d ₆ ) δ 150.51 (s), 149.39 (s). ESI-MS: m/z 874.4 [MH] ^-

1.22: Synthesis of Compound 159

The following process describes the synthesis of compound 159. Compound 159 was synthesized according to the process.

1.23: Synthesis of Compound 172

The following process describes the synthesis of compound 172. Compound 172 was synthesized according to the process.

1.24: General Synthesis Process 1

The above process describes general synthesis process 1, and compound 182 is synthesized according to the process.

Synthesis of compound 174:

Compound 173 (20.00 g, 133.21 mmol, 1.0 eq.) (Annegi, Lot.: RU3RRD3X) was dissolved in pyridine (200 mL). After complete dissolution, the solution was cooled in a 0°C bath. Acetic anhydride (68.00 g, 666.05 mmol, 5.0 eq.) was slowly added dropwise. After the addition was complete, the solution was slowly heated to room temperature and stirred overnight at room temperature until compound 173 reacted completely. The reaction solution was quenched with methanol, and an aqueous sodium bicarbonate solution was added. The reaction mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 45.65 g of crude compound 174. ESI-MS: m/z 319.1 [M+H] ⁺

Synthesis of compound 175:

Compound 174 (20.00 g, 62.84 mmol, 1.0 eq.) and N6-benzoyladenine (22.55 g, 94.26 mmol, 1.5 eq.) were added to a 500 mL three-necked round-bottom flask, and ultra-dry acetonitrile (200 mL) was added and stirred to dissolve. Then, BSA (38.35 g, 188.52 mmol, 3.0 eq.) was added. The reaction system was placed in an oil bath and heated to 80 °C, and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen protection. After the reaction was complete, the reaction system was placed in a 0 °C water bath and stirred for 30 minutes. TMSOTf (13.97 g, 62.84 mmol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition was complete, the reaction system was placed in an oil bath and slowly heated to 80 °C, and reacted overnight at this temperature. TLC and LCMS analysis showed that compound 174 reacted completely. The reaction mixture was removed from the oil bath and cooled to room temperature. The reaction was quenched by adding a saturated sodium bicarbonate aqueous solution. The mixture was extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give crude compound 175. The crude product was subjected to column chromatography (PE/EA = 1/5) to give compound 175 (20.22 g, 40.65 mmol, 64.69% yield). ESI-MS: m/z 498.2 [M+H] ⁺

Synthesis of compound 176:

Compound 175 (20.22 g, 40.65 mmol, 1.0 eq.) was added to a 500 mL round-bottom flask. THF (200 mL) was added to the reaction mixture, and the mixture was stirred until completely dissolved. A 5.4 mol/L sodium methoxide solution (24.84 mL, 134.15 mmol, 3.3 eq.) was added to the reaction mixture, and the mixture was stirred at room temperature until compound 175 was completely reacted. After the reaction was complete, the product precipitated. The reaction mixture was directly filtered, and the filter cake was washed twice with THF to give compound 176 (11.92 g, 32.10 mmol, 78.97% yield). ESI-MS: m/z 372.1 [M+H] ⁺

Synthesis of compound 177:

Compound 176 (11.92 g, 32.10 mmol, 1.0 eq.) was added to a 500 mL round-bottom flask. Pyridine (120 mL) was added to the reaction mixture, and the mixture was stirred until completely dissolved. Imidazole (5.46 g, 80.25 mmol, 2.5 eq.) and triphenylphosphine (12.63 g, 48.15 mmol, 1.5 eq.) were then added, followed by the slow addition of iodine (9.78 g, 38.52 mmol, 1.2 eq.). The mixture was stirred at room temperature until compound 176 was completely reacted. Imidazole (10.92 g, 160.50 mmol, 5.0 eq.) and TBSCl (14.51 g, 96.30 mmol, 3.0 eq.) were then added, and the mixture was stirred at room temperature until the intermediate was completely reacted. The reaction was quenched with methanol, and the mixture was extracted with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give crude compound 177. The crude product was subjected to column chromatography (PE/EA = 1/3) to give compound 177 (16.50 g, 23.25 mmol, 72.43% yield). ESI-MS: m/z 710.2 [M+H] ⁺

Synthesis of compound 178:

Compound 177 (16.50 g, 23.25 mmol, 1.0 eq.) was added to a 500 mL round-bottom flask, followed by the addition of THF (170 mL), and the mixture was stirred until completely dissolved. Then, DBU (7.08 g, 46.50 mmol, 2.0 eq.) was added, and the mixture was stirred at room temperature until compound 177 was completely dissolved. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude compound 178. The crude compound was subjected to column chromatography (PE/EA = 1/3) to give compound 178 (11.52 g, 19.80 mmol, 85.16% yield). ESI-MS: m/z 582.3 [M+H] ⁺

Synthesis of compound 179:

Compound 178 (11.52 g, 19.80 mmol, 1.0 eq.) was dissolved in ultradry acetonitrile (120 mL) and stirred. The mixture was then cooled in an ice bath. Et ₃ N·3HF (3.19 g, 19.80 mmol, 1.0 eq.) and NIS (5.35 g, 23.76 mmol, 1.2 eq.) were added. After addition, the system was stirred at room temperature. TLC analysis showed that compound 178 reacted completely. The system was directly filtered and concentrated to remove the solvent, yielding crude compound 179. Crude compound 179 was subjected to column chromatography (DCM/MeOH = 10/1) to obtain compound 179 (3.45 g, 6.91 mmol, 34.90% yield). ESI-MS: m/z 500.0 [M+H] ⁺

Synthesis of compound 180:

Compound 179 (3.45 g, 6.91 mmol, 1.0 eq.) was dissolved in methanol (40 mL) and stirred. Potassium carbonate (732 mg, 6.91 mmol, 1.0 eq.) was added, followed by three purgings with hydrogen gas. The reaction was stirred at room temperature. TLC analysis showed that compound 179 reacted completely. The system was directly filtered and concentrated to remove the solvent, yielding crude compound 180. Crude compound 180 was subjected to column chromatography (DCM/MeOH = 9/1) to obtain compound 180 (2.33 g, 6.24 mmol, 90.30% yield). ESI-MS: m/z 374.1 [M+H] ⁺

Synthesis of compound 181:

Compound 180 (2.33 g, 6.24 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DCE (30 mL) was added to the flask. The mixture was stirred until completely dissolved. DMTrCl (3.17 g, 9.36 mmol, 1.5 eq.), silver nitrate (1.06 g, 6.24 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (3.78 g, 31.20 mmol, 5.0 eq.) were added to the reaction mixture at room temperature and stirred until homogeneous. The reaction mixture was heated to 80 °C in an oil bath and allowed to react overnight. The reaction of compound 180 was confirmed to be complete by TLC and LCMS. The reaction mixture was then brought back to room temperature, and methanol was added to quench the reaction. The mixture was diluted with dichloromethane and filtered through diatomaceous earth to obtain a filtrate. The filter cake was washed twice with dichloromethane. The combined filtrates were concentrated under reduced pressure to obtain crude compound 181. The crude product was subjected to column chromatography (PE/EA = 1/6) to give compound 181 (3.42 g, 5.06 mmol, 81.09% yield). ESI-MS: m/z 674.2 [MH] ^-

Synthesis of compound 182:

The dried compound 181 (3.42 g, 5.06 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry dichloromethane (40 mL) was added and stirred until completely dissolved. DIPEA (1.31 g, 10.12 mmol, 2.0 eq.) and DMAP (123 mg, 1.01 mmol, 0.2 eq.) were added to the reaction mixture, and the mixture was stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under nitrogen protection. CEP-Cl (1.80 g, 7.59 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature, and the reaction was carried out at room temperature for 30–60 minutes until complete. The reaction was quenched by adding a saturated aqueous solution of sodium bicarbonate, and the reaction mixture was extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude compound 182. The crude product was purified by column chromatography (PE/EA = 1/4) to give compound 182 (3.60 g, 4.11 mmol, 81.25% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ10.16(s,1H),8.41(s,1H),8.16(s,1H),8.01–7.85(m,2H),7.56–7.40(m,3H),7.15–7.31(m,9H),6.83–6.62(m,4H),6.35(t,J＝9 .0Hz,1H),5.01(s,1H),3.79–3.66(m,3H),3.73(s,6H),2.97–2.73(m,2H),2.72–2.65(m,2H),1.12(s,3H),1.06(d,J＝12.0Hz,12H). ^31P NMR (162MHz, DMSO-d ₆ )δ152.81(s),152.70(s),151.75(s),151.61(s). ¹⁹ F NMR(377MHz, DMSO-d ₆ )δ-140.91(s),-140.78(s).ESI-MS: m/z 874.4[MH] ^-

1.25: General Synthesis Process 2

The above process describes general synthesis process 2, and compound 186 is synthesized according to the process.

The synthesis of compound 186 follows a similar synthetic route to that of compound 182, the difference being the orientation of the F atom at the 4' position. The NMR and mass spectrometry data for the synthesized compound 186 are as follows:

¹ H NMR (400MHz, DMSO-d ₆ )δ10.12(s,1H),8.43(s,1H),8.15(s,1H),8.02–7.83(m,2H),7.57–7.41(m,3H),7.13–7.32(m,9H),6.88–6.64(m,4H),6.31( t,J＝9.0Hz,1H),5.02(s,1H),3.78–3.61(m,9H),2.97–2.75(m,2H),2.71–2.61(m,2H),1.13(s,3H),1.05(d,J＝12.0Hz,12H). ^31P NMR (162MHz, DMSO-d ₆ )δ152.76(s),152.52(s),151.33(s),151.12(s). ¹⁹ F NMR(377MHz, DMSO-d ₆ )δ-140.86(s),-140.54(s).ESI-MS: m/z 874.4[MH] ^-

1.26: General Synthesis Process 3

The above process describes general synthesis process 3, and compound 191 is synthesized according to the process.

Synthesis of compound 187:

Compound 178 (10.00 g, 17.19 mmol, 1.0 eq.) was dissolved in ultradry methanol (100 mL) and stirred. Lead carbonate (9.19 g, 34.38 mmol, 1.0 eq.) was added, and the mixture was cooled in an ice bath. Iodine (8.73 g, 34.38 mmol, 1.0 eq.) was then added. After the addition was complete, the system was stirred at room temperature. The reaction was monitored by TLC. Compound 178 reacted completely. The system was directly filtered and concentrated to remove the solvent, yielding crude compound 187. Crude compound 187 was subjected to column chromatography (PE/EA = 1/3) to obtain compound 187 (4.23 g, 5.72 mmol, 33.28% yield). ESI-MS: m/z 740.2 [M+H] ⁺

Synthesis of compound 188:

Compound 187 (4.23 g, 5.72 mmol, 1.0 eq.) was dissolved in methanol (45 mL) and stirred. Potassium carbonate (606 mg, 5.72 mmol, 1.0 eq.) was added, followed by three purgings with hydrogen gas. The reaction was stirred at room temperature. TLC analysis showed that compound 187 reacted completely. The system was directly filtered and concentrated to remove the solvent, yielding crude compound 188. Crude compound 188 was subjected to column chromatography (PE/EA = 1/3) to obtain compound 188 (3.05 g, 4.97 mmol, 86.89% yield). ESI-MS: m/z 614.3 [M+H] ⁺

Synthesis of compound 189:

Compound 188 (3.05 g, 4.97 mmol, 1.0 eq.) was dissolved in methanol (30 mL) and stirred. Potassium fluoride (866 mg, 14.91 mmol, 3.0 eq.) was added, and the reaction was stirred at room temperature. TLC analysis showed that compound 188 reacted completely. The system was directly filtered and concentrated to remove the solvent, yielding crude compound 189. Crude compound 189 was subjected to column chromatography (DCM/MeOH = 10/1) to obtain compound 189 (1.55 g, 4.02 mmol, 80.89% yield). ESI-MS: m/z 386.1 [M+H] ⁺

Synthesis of compound 190:

Compound 189 (1.55 g, 4.02 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DCE (30 mL) was added to the flask. The mixture was stirred until completely dissolved. DMTrCl (2.04 g, 6.03 mmol, 1.5 eq.), silver nitrate (683 mg, 4.02 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (2.44 g, 20.10 mmol, 5.0 eq.) were added to the reaction mixture at room temperature and stirred until homogeneous. The reaction mixture was heated to 80 °C in an oil bath and allowed to react overnight. The reaction of compound 189 was confirmed to be complete by TLC and LCMS. The reaction mixture was then brought back to room temperature, and methanol was added to quench the reaction. The mixture was diluted with dichloromethane and filtered through diatomaceous earth to obtain a filtrate. The filter cake was washed twice with dichloromethane. The combined filtrates were concentrated under reduced pressure to obtain the crude compound 190. The crude product was subjected to column chromatography (PE/EA = 1/8) to give compound 190 (2.22 g, 3.23 mmol, 80.35% yield). ESI-MS: m/z 686.3 [MH ^]

Synthesis of compound 191:

The dried compound 190 (2.22 g, 3.23 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry dichloromethane (30 mL) was added and stirred until completely dissolved. DIPEA (835 mg, 6.46 mmol, 2.0 eq.) and DMAP (79 mg, 0.65 mmol, 0.2 eq.) were added to the reaction mixture, and the mixture was stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and the reaction was carried out under nitrogen protection. CEP-Cl (1.15 g, 4.85 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature, and the reaction was carried out at room temperature for 30–60 minutes until complete. The reaction was quenched by adding a saturated aqueous solution of sodium bicarbonate, and the reaction mixture was extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude compound 191. The crude product was purified by column chromatography (PE/EA = 1/6) to give compound 191 (2.35 g, 2.65 mmol, 82.04% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ10.12(s,1H),8.43(s,1H),8.15(s,1H),8.02–7.83(m,2H),7.57–7.41(m,3H),7.13–7.32(m,9H),6.86–6.61(m,4H),6.31(t,J＝9 .0Hz,1H),5.02(s,1H),3.78–3.61(m,9H),3.35(s,3H),2.97–2.75(m,2H),2.71–2.61(m,2H),1.13(s,3H),1.05(d,J＝12.0Hz,12H). ^31P NMR (162MHz, DMSO-d ₆ )δ152.73(s),151.35(s).ESI-MS: m/z 886.4[MH] ^-

1.27: General Synthesis Process 4

The above process describes general synthesis process 4, and compound 196 is synthesized according to the process.

The synthesis of compound 196 follows a similar synthetic route to that of compound 191, the difference being the orientation of the 4' methoxy group. The NMR and mass spectrometry data for the synthesis of compound 196 are as follows:

¹ H NMR (400MHz, DMSO-d ₆ )δ10.15(s,1H),8.45(s,1H),8.13(s,1H),8.03–7.85(m,2H),7.59–7.42(m,3H),7.12–7.31(m,9H),6.82–6.63(m,4H),6.32(t,J＝9 .0Hz,1H),5.03(s,1H),3.78–3.62(m,9H),3.37(s,3H),2.98–2.73(m,2H),2.70–2.60(m,2H),1.15(s,3H),1.03(d,J＝12.0Hz,12H). ^31P NMR (162MHz, DMSO-d ₆ )δ152.88(s),151.53(s).ESI-MS: m/z 886.4[MH] ^-

1.28: General Synthesis Process 5

The above process describes general synthesis process 5, and compound 201 is synthesized according to the process.

Synthesis of compound 197:

Compound 57 (10.00 g, 25.16 mmol, 1.0 eq.) was dissolved in ultradry acetonitrile (100 mL) and stirred. IBX (15.50 g, 55.35 mmol, 2.2 eq.) was added, and the system was purged with nitrogen and stirred at 80 °C. The reaction was monitored by TLC, and compound 57 was found to be completely reacted. The system was directly filtered and concentrated to remove the solvent, yielding crude compound 197 (10.56 g). Crude compound 197 was used directly in the next reaction. ESI-MS: m/z 396.1 [M+H] ⁺

Synthesis of compound 198:

The crude compound 197 (10.56 g) was dissolved and stirred in ultradry THF (150 mL), purged with nitrogen three times, and then cooled to -78 °C. Methyl magnesium bromide (4.50 g, 37.74 mmol, 1.5 eq.) was then slowly added. After the addition was complete, the mixture was heated to room temperature and stirred. TLC analysis showed that compound 197 reacted completely. The reaction was quenched by adding saturated ammonium chloride aqueous solution, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude compound 198. The crude compound was purified by column chromatography (PE/EA = 1/6) to give compound 198 (2.63 g, 6.39 mmol, 25.40% two-step yield). ESI-MS: m/z 412.4 [M+H] ⁺

Synthesis of compound 199:

Compound 198 (2.63 g, 6.39 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DCE (30 mL) was added to the reaction flask. The mixture was stirred until completely dissolved. DMTrCl (3.25 g, 9.59 mmol, 1.5 eq.), silver nitrate (1.09 g, 6.39 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (3.87 g, 31.90 mmol, 5.0 eq.) were added to the reaction mixture at room temperature and stirred until homogeneous. The reaction mixture was heated to 80 °C in an oil bath and allowed to react overnight. The reaction of compound 198 was confirmed to be complete by TLC and LCMS. The reaction mixture was brought back to room temperature, and methanol was added to quench the reaction. The mixture was diluted with dichloromethane and filtered through diatomaceous earth to obtain a filtrate. The filter cake was washed twice with dichloromethane. The combined filtrates were concentrated under reduced pressure to obtain crude compound 199. The crude product was subjected to column chromatography (PE/EA = 1/4) to give compound 199 (3.65 g, 5.11 mmol, 79.97% yield). ESI-MS: m/z 712.3 [MH ^]

Synthesis of compound 200:

Compound 199 (3.65 g, 5.11 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask. 40 mL of THF was added to the reaction mixture, and the mixture was stirred until completely dissolved. A 5.4 mol/L sodium methoxide solution (1.14 mL, 6.13 mmol, 1.2 eq.) was added to the reaction mixture, and the mixture was stirred at room temperature until compound 199 was completely reacted. After the reaction was complete, the product precipitated. The reaction mixture was directly filtered, and the filter cake was washed twice with THF to give compound 200 (3.02 g, 4.50 mmol, 88.06% yield). ESI-MS: m/z 670.3 [MH] ^-

Synthesis of compound 201:

The dried compound 200 (3.02 g, 4.50 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry dichloromethane (30 mL) was added and stirred until completely dissolved. DIPEA (1.09 g, 9.00 mmol, 2.0 eq.) and DMAP (110 mg, 0.90 mmol, 0.2 eq.) were added to the reaction mixture, and the mixture was stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under nitrogen protection. Compound CEP-Cl (1.60 g, 6.75 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature, and the reaction was carried out at room temperature for 30–60 minutes until complete. The reaction was quenched by adding a saturated aqueous solution of sodium bicarbonate, and the reaction mixture was extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude compound 201. The crude product was purified by column chromatography (PE/EA = 1/4) to give compound 201 (3.35 g, 3.84 mmol, 85.33% yield). ¹ H NMR (400MHz, DMSO-d ₆ )δ10.11(s,1H),8.42(s,1H),8.12(s,1H),8.02–7.89(m,2H),7.58–7.41(m,3H),7.11–7.30(m,9H),6.85–6.59(m,4H),6.33(t,J＝9 .0Hz,1H),5.01(s,1H),3.79–3.61(m,9H),2.97–2.72(m,2H),2.71–2.61(m,2H),1.22(s,3H),1.13(s,3H),1.03(d,J＝12.0Hz,12H). ^31P NMR (162MHz, DMSO-d ₆ )δ152.77(s),151.65(s).ESI-MS:m/z 870.4[MH] ^-

1.28: General Synthesis Process 6

The above process describes general synthesis process 6, and compound 211 is synthesized according to the process.

The synthesis of compound 211 follows a similar synthetic route to that of compound 201, the difference being the orientation of the 4' methoxy group. The NMR and mass spectrometry data for the synthesis of compound 211 are as follows:

¹ H NMR (400MHz, DMSO-d ₆ )δ10.12(s,1H),8.43(s,1H),8.14(s,1H),8.03–7.85(m,2H),7.59–7.42(m,3H),7.12–7.32(m,9H),6.79–6.63(m,4H),6.33(t,J＝9 .0Hz,1H),5.02(s,1H),3.79–3.62(m,9H),2.99–2.72(m,2H),2.72–2.61(m,2H),1.23(s,3H),1.14(s,3H),1.02(d,J＝12.0Hz,12H). ^31P NMR (162MHz, DMSO-d ₆ )δ152.79(s),151.63(s).ESI-MS:m/z 870.4[MH] ^-

In the following examples, monomers were selected from Table 1 below for use in the synthesis of oligonucleotides.

Table 1. Names and structures of compounds used in oligonucleotide synthesis






Note: R in Table 1 represents methyl or C2-C30 alkyl.

Example 2: Oligonucleotide Synthesis

The siRNA was synthesized using the conventional phosphoramide solid-phase method. When synthesizing nucleotides modified at various positions in the SS and AS chains, the original nucleotides in the parent sequences shown in Table 2A were replaced with the phosphoramide monomers synthesized above.

The synthesis process is briefly described as follows: Using an LK-48E synthesizer (Lingkun), starting with a Universal CPG carrier, nucleoside phosphoramide monomers were linked one by one according to the synthesis program. In addition to the OT3-A, U, G, and C series compounds described above, the remaining nucleoside monomer raw materials, such as 2'-F RNA and 2'-O-methyl RNA, were purchased from Shanghai Zhaowei. 5'-Ethylthio-1H-tetrazole (ETT) was used as the activator (0.6M acetonitrile solution), 0.22M PADS dissolved in a 1:1 volume ratio of trimethylpyridine (Suzhou Kelama) solution was used as the sulfiding agent, and iodopyridine/water solution (Kelama) was used as the oxidizing agent.

After solid-phase synthesis, the oligonucleotides were cleaved from the solid support and soaked in a 3:1 solution of 28% ammonia and ethanol at 50°C for 16 hours. After centrifugation, the supernatant was transferred to another centrifuge tube, concentrated, and evaporated to dryness. Purification was then performed using C18 reversed-phase chromatography with 0.1M TEAA and acetonitrile as the mobile phase, and DMTr was removed using 3% trifluoroacetic acid solution. The target oligonucleotides were collected, lyophilized, identified as the target product by LCMS, and then quantified by UV (260 nm).

The obtained single-stranded oligonucleotides were annealed according to the equimolar ratio and complementary pairing. The resulting double-stranded siRNA (Table 2B) was dissolved in 1xPBS and adjusted to the required concentration for the experiment.

Table 2A. Unmodified dsRNA

Table 2B. Modified dsRNA

Table 2C lists further details of the molecules in Table 2B, where the structure of each synthesized molecule is defined by the macromolecular hierarchical editing language (HELM) (Zhang et al., Chem. Inf. Model. 2012; 52(10):2796-2806). The following HELM annotations are used:

[mR](A) is a 2′-O-methylRNA adenine nucleoside.

[mR](C) is a 2'-O-methylRNA cytosine nucleoside.

[mR](G) is a 2'-O-methylRNA-guanine nucleoside.

[mR](U) is a 2'-O-methylRNA uracil nucleoside.

[dR](T) is a DNA thymidine nucleoside.

[fR](A) is a 2′-fluoroRNA adenine nucleoside.

[fR](C) is a 2′-fluoroRNA cytosine nucleoside.

[fR](G) is a 2′-fluoroRNA guanine nucleoside.

[fR](U) is a 2′-fluoroRNA uracil nucleoside.

[TNA](A) is a threonine adenosine nucleoside.

[TNA](T) is a threonine thymidine nucleoside.

[TNA](G) is a threonine guanine nucleoside.

[TNA](C) is a threonine cytosine nucleoside.

[TNA](U) is a threonine uridine nucleoside.

[Rf4TNA](A) is a 4'-(R)-fluorothreonine adenosine nucleoside.

[Rf4TNA](T) is a 4'-(R)-fluorothreonine thymidine nucleoside.

[Rf4TNA](G) is a 4'-(R)-fluorothreonine guanine nucleoside.

[Rf4TNA](C) is a 4'-(R)-fluorothreonine cytosine nucleoside.

[Rf4TNA](U) is a 4'-(R)-fluorothreonine uridine nucleoside.

[Smo4TNA](A) is a 4'-(S)-O-methylthreonine adenosine nucleoside.

[Smo4TNA](T) is a 4'-(S)-O-methylthreonine thymidine nucleoside.

[Smo4TNA](G) is a 4'-(S)-O-methylthreonine guanine nucleoside.

[Smo4TNA](C) is a 4'-(S)-O-methylthreonine cytosine nucleoside.

[Smo4TNA](U) is a 4'-(S)-O-methylthreonine uridine nucleoside.

[Rmo4TNA](A) is a 4'-(R)-O-methylthreonine adenosine nucleoside.

[Rmo4TNA](T) is a 4'-(R)-O-methylthreonine thymidine nucleoside.

[Rmo4TNA](G) is a 4'-(R)-O-methylthreonine guanine nucleoside.

[Rmo4TNA](C) is a 4'-(R)-O-methylthreononucleotide cytosine nucleoside.

[Rmo4TNA](U) is a 4'-(R)-O-methylthreononucleotide uracil.

[Rm4TNA](A) is a 4'-(R)-methylthreonine adenosine nucleoside.

[Rm4TNA](T) is a 4'-(R)-methylthreonine thymidine nucleoside.

[Rm4TNA](G) is a 4'-(R)-methylthreonine guanine nucleoside.

[Rm4TNA](C) is a 4'-(R)-methylthreonine cytosine nucleoside.

[Rm4TNA](U) is a 4'-(R)-methylthreonine uridine nucleoside.

[Rfm4TNA](A) is a 4'-(R) _-CH2F -threonine adenosine nucleoside.

[Rfm4TNA](T) is a 4'-(R) _-CH2F -threonine thymidine nucleoside.

[Rfm4TNA](G) is a 4'-(R) _-CH2F -threonoguanine nucleoside.

[Rfm4TNA](C) is a 4'-(R) _-CH2F -threononucleotide cytosine nucleoside

[Rfm4TNA](U) is a 4'-(R) _-CH2F -threononucleotide uracil.

[Rmom4TNA](A) is a 4'-(R) _-CH2OMe -threonine adenosine nucleoside.

[Rmom4TNA](T) is a 4'-(R) _-CH2OMe -threonine thymidine nucleoside.

[Rmom4TNA](G) is a 4'-(R) _-CH2OMe -threonine guanine nucleoside.

[Rmom4TNA](C) is a 4'-(R) _-CH2OMe -threonine cytosine nucleoside.

[Rmom4TNA](U) is a 4'-(R) _-CH2OMe -threonucleic acid uracil nucleoside.

[Rhm4TNA](A) is a 4'-(R) _-CH2OH -threonine adenosine nucleoside.

[Rhm4TNA](T) is a 4'-(R) _-CH2OH -threonine thymidine.

[Rhm4TNA](G) is a 4'-(R) _-CH2OH -threonine guanine nucleoside.

[Rhm4TNA](C) is a 4'-(R) _-CH2OH -threonine cytosine nucleoside.

[Rhm4TNA](U) is a 4'-(R) _-CH2OH -threonine uridine nucleoside.

[Sm4TNA](A) is a 4'-(S)-methylthreonine adenosine nucleoside.

[Sm4TNA](T) is a 4'-(S)-methylthreonine thymidine nucleoside.

[Sm4TNA](G) is a 4'-(S)-methylthreonine guanine nucleoside.

[Sm4TNA](C) is a 4'-(S)-methylthreonine cytosine nucleoside.

[Sm4TNA](U) is a 4'-(S)-methylthreonine uridine nucleoside.

[Sfm4TNA](A) is a 4'-(S) _-CH2F -threononucleotide adenosine.

[Sfm4TNA](T) is a 4'-(S) _-CH2F -threonine thymidine nucleoside.

[Sfm4TNA](G) is a 4'-(S) _-CH2F -threonoguanine nucleoside.

[Sfm4TNA](C) is a 4'-(S) _-CH2F -threononucleotide cytosine nucleoside

[Sfm4TNA](U) is a 4'-(S) _-CH2F -threononucleotide uracil.

[Smom4TNA](A) is a 4'-(S) _-CH2OMe -threonine adenosine nucleoside.

[Smom4TNA](T) is a 4'-(S) _-CH2OMe -threonine thymidine nucleoside.

[Smom4TNA](G) is a 4'-(S) _-CH2OMe -threonine guanine nucleoside.

[Smom4TNA](C) is a 4'-(S) _-CH2OMe -threonine cytosine nucleoside.

[Smom4TNA](U) is a 4'-(S) _-CH2OMe -threonucleic acid uracil nucleoside.

[Rm4Rhm3TNA](A) is a 4'-(R) _-CH3-3 '-(R) _-CH2OH -threonine nucleoside.

[Rm4Rhm3TNA](T) is a 4'-(R) _-CH3-3 '-(R) _-CH2OH -threonine thymidine.

[Rm4Rhm3TNA](G) is a 4'-(R) _-CH3-3 '-(R) _-CH2OH -threonine guanine nucleoside.

[Rm4Rhm3TNA](C) is a 4'-(R) _-CH3-3 '-(R) _-CH2OH -threonine cytosine nucleoside

[Rm4Rhm3TNA](U) is a 4'-(R) _-CH3-3 '-(R) _-CH2OH -threonine uridine nucleoside.

[Rmo4Rhm3TNA](A) is a 4'-(R)-O-methyl-3'-(R) _-CH2OH -threononucleotide adenine nucleoside

[Rmo4Rhm3TNA](T) is a 4'-(R)-O-methyl-3'-(R) _-CH2OH -threonine thymidine.

[Rmo4Rhm3TNA](G) is a 4'-(R)-O-methyl-3'-(R) _-CH2OH -threonoguanine nucleoside.

[Rmo4Rhm3TNA](C) is a 4'-(R)-O-methyl-3'-(R) _-CH2OH -threononucleotide cytosine nucleoside

[Rmo4Rhm3TNA](U) is a 4'-(R)-O-methyl-3'-(R) _-CH2OH -threononucleotide uracil nucleoside

[f4m4STNA](A) is a 4’-F-4’-Me-(S)-threonine adenosine nucleoside.

[f4m4STNA](T) is a 4’-F-4’-Me-(S)-threonine thymidine nucleoside.

[f4m4STNA](G) is a 4’-F-4’-Me-(S)-threonine guanine nucleoside.

[f4m4STNA](C) is a 4’-F-4’-Me-(S)-threonine cytosine nucleoside.

[f4m4STNA](U) is a 4’-F-4’-Me-(S)-threonine uridine nucleoside.

[f4m4RTNA](A) is a 4’-F-4’-Me-(R)-threonine adenosine nucleoside.

[f4m4RTNA](T) is a 4'-F-4'-Me-(R)-threonine thymidine nucleoside.

[f4m4RTNA](G) is a 4’-F-4’-Me-(R)-threonine guanine nucleoside.

[f4m4RTNA](C) is a 4’-F-4’-Me-(R)-threonine cytosine nucleoside.

[f4m4RTNA](U) is a 4’-F-4’-Me-(R)-threonine uridine nucleoside.

[mo4m4RTNA](A) is a 4’-OMe-4’-Me-(R)-threonine adenosine nucleoside.

[mo4m4RTNA](T) is a 4’-OMe-4’-Me-(R)-threonine thymidine nucleoside.

[mo4m4RTNA](G) is a 4’-OMe-4’-Me-(R)-threonine guanine nucleoside.

[mo4m4RTNA](C) is a 4’-OMe-4’-Me-(R)-threonine cytosine nucleoside.

[mo4m4RTNA](U) is a 4’-OMe-4’-Me-(R)-threonine uridine nucleoside.

[mo4m4STNA](A) is a 4’-OMe-4’-Me-(S)-threonine adenosine nucleoside.

[mo4m4STNA](T) is a 4’-OMe-4’-Me-(S)-threonine thymidine nucleoside.

[mo4m4STNA](G) is a 4’-OMe-4’-Me-(S)-threonine guanine nucleoside.

[mo4m4STNA](C) is a 4’-OMe-4’-Me-(S)-threonine cytosine nucleoside.

[mo4m4STNA](U) is a 4’-OMe-4’-Me-(S)-threonine uridine nucleoside.

[Rm4Sm3TNA](A) is a 4’-(R)-Me-3’-(S)-Me-threonine adenosine nucleoside.

[Rm4Sm3TNA](T) is a 4’-(R)-Me-3’-(S)-Me-threonine thymidine.

[Rm4Sm3TNA](G) is a 4’-(R)-Me-3’-(S)-Me-threonine guanine nucleoside.

[Rm4Sm3TNA](C) is a 4’-(R)-Me-3’-(S)-Me-threonine cytosine nucleoside.

[Rm4Sm3TNA](U) is a 4’-(R)-Me-3’-(S)-Me-threonine uridine nucleoside.

[Sm4Sm3TNA](A) is a 4'-(S)-Me-3'-(S)-Me-threonine adenosine nucleoside.

[Sm4Sm3TNA](T) is a 4'-(S)-Me-3'-(S)-Me-threonine thymidine.

[Sm4Sm3TNA](G) is a 4’-(S)-Me-3’-(S)-Me-threonine guanine nucleoside.

[Sm4Sm3TNA](C) is a 4'-(S)-Me-3'-(S)-Me-threonine cytosine nucleoside.

[Sm4Sm3TNA](U) is a 4’-(S)-Me-3’-(S)-Me-threonine uridine nucleoside.

[sP] is the internucleotide bond of thiophosphate ester.

[P] represents the nucleotide inter-bond of the phosphodiester.

Further information about HELM and its open-source tools can be found at www.pistoiaalliance.org/helm-tools/ (accessed August 26, 2022).

Table 2C. Modified dsRNA

Example 3: Detection of in vitro target and off-target activities of siRNA compounds

3.1 In vitro reporter gene assay

3.1.1 Cell Culture and Transfection

HEK293A cells (Chinese Academy of Sciences Cell Bank, catalog number SCSP- ₅₀₉₄ ) were cultured in DMEM (Gibco, cat#11885-084) supplemented with 10% fetal bovine serum (FBS) until near-confluence. The cells were then digested and resuspended in cell seeding medium (DMEM + 10% FBS), and seeded at 10,000 cells/well/150 μL into 96-well cell culture plates (Costar, cat#3599) and cultured overnight at 37°C and 5% CO2 _. Cells were transfected using the siRNA and psi-CHCECK2 plasmid listed in Table 3, where the target sequence (HAO1) in the psi-CHCECK2 plasmid is as follows:

On-target: ATAAACAAGGTTTGACATCAATCTAGCTATATCTTTAAGAATGATAAACGAATGTGAAAGTCATCGACAAGACATTGGTGAGGAAAAATCCTTTGGCCGTTTCCAAGATCTGACAGTGCA (SEQ ID NO: 6)

Off-target: ATAAACAAGGTTTGACATCAATCTAGCTATATCTTTAAGAATGATAAACTCATCGACAATAATATTACATAAAATAAAACATCGACAATAATATTACATAAAAAACATCGACAATAATATTACATAAATAAAACATCGACAAGACATTGGTGAGGAAAAATCCTTTGGCCGTTTCCAAGATCTGACAGTGCA(SEQ ID NO:7)

The target sequence (TTR) in the psi-CHCECK2 plasmid is as follows:

On-target: ATAAACAAGGTTTGACATCAATCTAGCTATATCTTTAAGAATGATAAACTAAACAGTGTTCTTGCTCTATAAGACATTGGTGAGGAAAAATCCTTTGGCCGTTTCCAAGATCTGACAGTGCA (SEQ ID NO: 8)

Off-target: ATAAACAAGGTTTGACATCAATCTAGCTATATCTTTAAGAATGATAAACTGCTCTATAATAATATTACATAAATA AAAGCTCTATAATAATATTACATAAATAAAAGCTCTATAATAATATTACATAAATAAAAGCTCTATAAGACATTGGTGAGGAAAAATCCTTTGGCCGTTTCCAAGATCTGACAGTGCA(SEQ ID NO:9)

Add 5 μL of double-stranded siRNA and 100 ng of psi-CHECK2 plasmid to each well; mix 24.6 μL of Opti-MEM and 0.4 μL of Lipofectamine 2000 (Invitrogen, cat#11668500), incubate at room temperature for 20 minutes, then add the mixture to the cells, 30 μL per well, and incubate the transfected cells overnight at 37°C in 5% _CO2 .

3.2 Dual-luciferase activity assay

Following the manufacturer's instructions, cells were harvested 24 hours after transfection for a dual-luciferase assay (Promega, cat#E2940). First, the culture medium was removed from the cells. Then, 50 μL of culture medium and 50 μL of luciferase reagent were added to each well, mixed for 10 min, and the firefly luciferase activity was measured on a Spark (Tecan) instrument. Next, 50 μL of Renida luciferase substrate was added to each well, incubated for 10–15 min, and luminescence was measured again to determine the Renida luciferase activity.

siRNA activity = (sample's Renal luciferase value / sample's firefly luciferase value) / (MOCK's Renal luciferase value / MOCK's firefly luciferase value). All transfections were performed in triplicate (n = 3).

Table 3. Experimental results of in vitro testing of siRNA compound activity against targets using psi-CHECK2 plasmid

Table 4. Experimental results of in vitro testing of the off-target activity of siRNA compounds using the psi-CHECK2 plasmid.

As can be seen from the results in Tables 3 and 4, using modified nucleosides in the seed region of the antisense strand of the dsRNA reagent can provide reduced off-target binding in vitro.

Example 4: Synthesis of siRNA sequence for in vivo evaluation

The synthesis of siRNA is no different from the usual phosphoramide solid-phase synthesis method. When synthesizing nucleotides modified at various positions of the SS and AS chains, the original nucleotides in the parent sequence shown in Table 2A are replaced with the phosphoramide monomers synthesized above.

The synthesis process is briefly described as follows: Using an LK-48E synthesizer (Lingkun), starting with a Universal CPG (Dinaxinke) carrier, nucleoside phosphoramide monomers were linked one by one according to the synthesis program. Except for the OT series compounds described above, the remaining nucleoside monomer raw materials, such as 2'-F RNA and 2'-O-methyl RNA, were purchased from Shanghai Zhaowei, and L96 was purchased from Tangzhi Pharmaceutical. 5'-Ethylthio-1H-tetrazole (ETT) was used as the activator (0.6M acetonitrile solution), 0.22M PADS dissolved in a 1:1 volume ratio of trimethylpyridine (Suzhou Kelema) solution was used as the sulfiding agent, and iodopyridine/water solution (Kelema) was used as the oxidizing agent.

After solid-phase synthesis, the oligonucleotides were cleaved from the solid support and soaked in a 3:1 solution of 28% ammonia and ethanol at 50°C for 16 hours. After centrifugation, the supernatant was transferred to another centrifuge tube, concentrated, and evaporated to dryness. Purification was then performed using C18 reversed-phase chromatography with 0.1M TEAA and acetonitrile as the mobile phase, and DMTr was removed using 3% trifluoroacetic acid solution. The target oligonucleotides were collected, lyophilized, identified as the target product by LCMS, and then quantified by UV (260 nm).

The obtained single-stranded oligonucleotides and sodium acetate were ultrafiltered and salt-replaced using a 3KD ultrafiltration tube. According to the equimolar ratio, they were paired complementary and annealed. Finally, the resulting double-stranded siRNA-L96 conjugate (Table 5) was dissolved in water and adjusted to the required concentration for the experiment.

Table 5. Double-stranded siRNA-L96 conjugates

Note: In Table 5, "m" represents 2'-OMe modification, "f" represents 2'-F modification, "s" represents PS backbone modification, "L96" represents liver-targeting ligand, "OT3" represents 4'-(R)-Me-TNA modification, "OT4" represents 4'- _CH2F -TNA modification, and "OT5" represents 4'- _CH2 The modifications are as follows: "OT10" is modified with 4'-(S)-Me-TNA, "OT25" is modified with 4'-F-4'-Me-(S)-TNA, "OT26" is modified with 4'-F-4'-Me-(R)-TNA, "OT27" is modified with 4'-OMe-4'-Me-(R)-TNA, "OT28" is modified with 4'-OMe-4'-Me-(S)-TNA, "OT29" is modified with 4'-(R)-Me-3'-(S)-Me-TNA, and "OT30" is modified with 4'-(S)-Me-3'-(S)-Me-TNA. For details of the modifications, please refer to the abbreviation table of this invention.

Table 6 lists further details of the molecules in Table 5, with the structure of each synthetic molecule defined by the macromolecular hierarchical editing language (HELM).

Table 6. Double-stranded siRNA-L96 conjugates

Example 5: In vivo activity evaluation of siRNA duplexes containing modified threononucleotides

The in vivo activity of compounds BPR3M01-23087-L96～BPR3M01-23092-L96 and BPR3M01-23105-L96～BPR3M01-23110-L96 (see Table 5) against TTR target mRNA was evaluated using wild-type C57BL/6 mice (Speford (Beijing) Biotechnology Co., Ltd.).

Six- to eight-week-old C57BL/6 mice were administered a single subcutaneous injection of the compound at doses of 0.5 mg/kg or 1 mg/kg. Orbital blood was collected in EP tubes before administration and at days 7, 14, 21, 28, 35, 42, 56, 63, and 70 post-administration. After the blood samples were allowed to stand at room temperature for two hours, they were centrifuged at 5500 rpm for 10 min at 4°C to separate and collect serum for the determination of TTR levels in the animal serum.

Serum TTR levels were detected by enzyme-linked immunosorbent assay (ELISA). The mouse Prealbumin ELISA kit (Abcam, catalog number: ab282297) was used for ELISA detection, and all samples were tested according to the kit instructions. Absorbance at 450 nm was read on a Tecan Spark microplate reader, and the data from the standards (from the aforementioned ELISA kit) were fitted to a parametric standard curve to determine serum TTR protein levels (in μg/mL). The protein content of each animal was compared with its corresponding pre-drug serum protein content to determine the percentage of TTR remaining relative to pre-drug levels.

ELISA results (Figure 1) showed that the modified threonucleotide-containing siRNA duplexes BPR3M01-23105-L96, BPR3M01-23107-L96, and BPR3M01-23109-L96 significantly reduced serum TTR levels. They exhibited similar in vivo activity to the unmodified threonucleotide-free siRNA duplex BPR3M01-23087-L96, indicating that these modified nucleoside compounds can maintain the silencing activity of siRNA sequences in vivo.

The foregoing describes exemplary embodiments of the present invention. Those skilled in the art should understand that these disclosures are merely exemplary, and various other substitutions, adaptations, and modifications can be made within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments listed herein.

Claims (30)

Compound of formula (I)
in, R ₁ is selected from halogen, alkyl, -O-alkyl, -S-alkyl and -NH-alkyl, wherein the alkyl is optionally substituted by one or more substituents selected from the following: hydroxyl, cyano, halogen, -O-alkyl, -S-alkyl, -NH ₂ , -NH-alkyl and -N(alkyl) ₂ ; _R2 is selected from H, halogens, alkyl groups, and -O-alkyl groups; R ₃ is selected from -O-DMTr or -CH ₂ -X-DMTr, where X is O, S or NH, and DMTr represents 4,4'-dimethoxytriphenylmethyl; Where Base is a natural or non-natural base, or a protected natural or non-natural base. The compound according to claim 1 is selected from formula (Ia) and formula (Ib).
_R1 , _R2 , _R3 and Base are defined as in claim 1. The compound according to claim 1 or 2, wherein the alkyl group is selected from _C1 - _C30 alkyl groups, such as _C1 - _C10 alkyl groups, _C1 - _C6 alkyl groups, _C1 - _C4 alkyl groups, and the alkyl group is straight-chain or branched. The compound according to any one of claims 1 to 3, wherein _R1 is selected from halogen, _C1 - _C6 alkyl, -O-( _C1 - _C6 alkyl), -S-( _C1 - _C6 alkyl), and -NH-( _C1 - _C6 alkyl), wherein the _C1 - _C6 alkyl is optionally substituted by one or more substituents selected from: hydroxyl, cyano, halogen, -O-( _C1 - _C6 alkyl), -S-( _C1 - _C6 alkyl), _-NH2 , -NH-( _C1 - _C6 alkyl), and -N( _C1 - _C6 alkyl) ₂ ; _R2 is selected from H, halogens, _C1 - _C6 alkyl groups and -O-( _C1 - _C6 alkyl groups); R ₃ is selected from -O-DMTr or -CH ₂ -X-DMTr, where X is O, S or NH, and DMTr represents 4,4'-dimethoxytriphenylmethyl; Where Base is a natural or non-natural base, or a protected natural or non-natural base. The compound according to any one of claims 1 to 4, wherein _R1 is selected from halogens, _C1 - _C6 alkyl groups, and -O-( _C1 - _C6 alkyl groups), wherein the _C1 - _C6 alkyl group is optionally substituted with a substituent selected from: hydroxyl, halogen, -O-( _C1 - _C6 alkyl), -S-( _C1 - _C6 alkyl), _-NH2 , -NH-( _C1 - _C6 alkyl), and -N( _C1 - _C6 alkyl) ₂ ; preferably, _R1 is selected from F, methoxy, and methyl groups optionally substituted with hydroxyl, halogen, -O-( _C1 - _C6 alkyl), -S-( _C1 - _C6 alkyl), and -NH-( _C1 - _C6 alkyl); more preferably, _R1 is selected from F, _-CH3 , _-OCH3 , _-CH2 -OH, _-CH2F , _-CH2 - _OCH3 , and _-CH2 - _SCH3. -CH2 _- NH( _CH3 ). The compound according to any one of claims 1 to 5, wherein _R2 is selected from H, _C1 - _C6 alkyl and -O-( _C1 - _C6 alkyl); preferably, _R2 is selected from H, methyl and methoxy; more preferably, _R2 is H. The compound according to any one of claims 1 to 6, wherein neither _R1 nor _R2 is H; for example, _R1 is selected from halogens, _C1 - _C6 alkyl groups, and -O-( _C1 - _C6 alkyl), and _R2 is selected from _C1 - _C6 alkyl groups and -O-( _C1 - _C6 alkyl), wherein the _C1 - _C6 alkyl group is optionally substituted with a substituent selected from: hydroxyl, halogen, -O-( _C1 - _C6 alkyl), -S-( _C1 - _C6 alkyl), _-NH2 , -NH-( _C1 - _C6 alkyl), and -N( _C1 - _C6 alkyl) ₂ ; preferably, _R1 is selected from F, methoxy, and methyl groups optionally substituted with hydroxyl, halogen, -O-( _C1 - _C6 alkyl), -S-( _C1 - _C6 alkyl), and -NH-( _C1 - _C6 alkyl), and _R2 is selected from _C1 -C6 alkyl. _6- alkyl and -O-( _C1 - _C6alkyl ); most preferably, _R1 is selected from F, _-CH3 , _-OCH3 , _-CH2- OH, _-CH2F , _-CH2 - _OCH3 , _-CH2 - _SCH3 , _-CH2- NH( _CH3 ), and _R2 is selected from methyl and methoxy; for example, _R1 is selected from F, _-CH3 , _-OCH3 , and _R2 is selected from methyl. The compound according to any one of claims 1 to 7, wherein _R3 is selected from -O-DMTr or _-CH2 -O-DMTr. The compound according to any one of claims 1 to 8, wherein the natural base is selected from natural bases A, U, C, G, and T; the non-natural base is selected from non-natural purine bases and non-natural pyrimidine bases; for example, the non-natural purine base is independently selected from the following bases:
The non-natural pyrimidine bases are independently selected from the following bases:
The compound according to any one of claims 1 to 9, wherein the compound is selected from:

R is methyl or C2-C30 alkyl, Base is as defined in claim 1, preferably selected from natural bases A, U, G, C, T or protected bases A, U, G, C, T; and the stereochemistry is R or S, and for any unspecified chiral center it is a combination of R and S. Preferably, the compound is selected from:

Wherein Base is as defined in claim 1, preferably Base is selected from A, U, G, C, T or protected A, U, C, G, T. Compound of formula (II)
in: R ₁ is selected from halogen, alkyl, -O-alkyl, -S-alkyl and -NH-alkyl, wherein the alkyl is optionally substituted by one or more substituents selected from the following: hydroxyl, cyano, halogen, -O-alkyl, -S-alkyl, -NH ₂ , -NH-alkyl and -N(alkyl) ₂ ; R ₃ is selected from -O-DMTr or -CH ₂ -X-DMTr, where X is O, S or NH, and DMTr represents 4,4'-dimethoxytriphenylmethyl; R ₄ is selected from halogens, alkyl groups, and -O-alkyl groups; Where Base is a natural or non-natural base, or a protected natural or non-natural base. The compound according to claim 11 is selected from compounds of formula (IIa) and (IIb).
_R1 , _R3 , _R4 and Base are defined as in claim 11. The compound according to claim 11 or 12, wherein the alkyl group is selected from _C1 - _C30 alkyl groups, such as _C1 - _C10 alkyl groups, _C1 - _C6 alkyl groups, _C1 - _C4 alkyl groups, and the alkyl group is straight-chain or branched. The compound according to any one of claims 11 to 13, wherein _R1 is selected from halogen, _C1 - _C6 alkyl, -O-( _C1 - _C6 alkyl), -S-( _C1 - _C6 alkyl), and -NH-( _C1 - _C6 alkyl), wherein the _C1 - _C6 alkyl is optionally substituted by one or more substituents selected from: hydroxyl, cyano, halogen, -O-( _C1 - _C6 alkyl), -S-( _C1 - _C6 alkyl), _-NH2 , -NH-( _C1 - _C6 alkyl), and -N( _C1 - _C6 alkyl) ₂ ; R ₃ is selected from -O-DMTr or -CH ₂ -X-DMTr, where X is O, S or NH, and DMTr represents 4,4'-dimethoxytriphenylmethyl; _R4 is selected from halogens, _C1 - _C6 alkyl groups, and -O-( _C1 - _C6 alkyl groups); Where Base is a natural or non-natural base, or a protected natural or non-natural base. The compound according to any one of claims 11 to 14, wherein _R1 is selected from halogens, _C1 - _C6 alkyl groups, and -O-( _C1 - _C6 alkyl groups), wherein the _C1 - _C6 alkyl group is optionally substituted with a substituent selected from the following: hydroxyl, halogen, -O-( _C1 - _C6 alkyl), -S-( _C1 - _C6 alkyl), _-NH2 , -NH-( _C1 - _C6 alkyl), and -N( _C1 - _C6 alkyl) ₂ ; preferably, _R1 is selected from F, methoxy, and methyl groups optionally substituted with hydroxyl, halogen, -O-( _C1 - _C6 alkyl), -S-( _C1 - _C6 alkyl), and -NH-( _C1 - _C6 alkyl); more preferably, _R1 is selected from _-CH3 . The compound according to any one of claims 11 to 15, wherein _R4 is selected from _C1 - _C6 alkyl and -O-( _C1 - _C6 alkyl); preferably, _R4 is selected from methyl. The compound according to any one of claims 11 to 16, wherein _R3 is selected from -O-DMTr or _-CH2 -O-DMTr. The compound according to any one of claims 11 to 17, wherein Base is selected from natural bases A, U, C, G, T or protected natural bases A, U, C, G, T; or Base is selected from non-natural purine bases and non-natural pyrimidine bases as defined in claim 9. The compound according to any one of claims 11 to 18, wherein the compound is
Wherein Base is defined as in claim 11, preferably Base is selected from natural bases A, U, G, C, T or protected bases A, U, C, G, T. A double-stranded RNA molecule comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand and the antisense strand each have 14-40 nucleotides, for example, 15-30, for example, 17-25, 19-23 nucleotides, respectively, wherein the antisense strand is sufficiently complementary to a target sequence to mediate RNA interference, wherein the antisense strand comprises at least one compound of any one of claims 1 to 19 at the nucleotide positions 2-8 (e.g., 5-8) of the 5' region (counting from the 5' end). According to the double-stranded RNA molecule of claim 20, wherein the sense strand further comprises a ligand attached to either end of the sense strand, the ligand being covalently linked to the sense strand via a linker, for example, the ligand being an asialic acid glycoprotein receptor (ASGPR) ligand, for example, the ASGPR ligand comprising one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent linker. The double-stranded RNA molecule according to claim 20 or 21, wherein the sense strand has a modified nucleoside, such as DNA, 2’-OMe, 2’-F, 2’-O-MOE, or TNA, at a position where it pairs with the nucleotide position of the compound of any one of claims 1 to 19 on the antisense strand. The double-stranded RNA molecule according to any one of claims 20-22, wherein substantially all nucleotides of the antisense strand and/or the sense strand are modified nucleotides, preferably, the sense strand and/or antisense strand comprises at least one modified nucleotide selected from the group consisting of 2’-O-methyl modified nucleotides, 2’-fluoro modified nucleotides, 2’-O-methoxyethyl modified nucleotides, and deoxyribonucleotides. The double-stranded RNA molecule according to any one of claims 20-23, wherein the double-stranded RNA molecule is a dsRNA molecule that inhibits the expression of the transthyretin (TTR) gene, wherein the sense strand comprises at least 15 consecutive nucleotides, such as 15, 16, 17, 18, 19, 20, or 21, that differ from the nucleotide sequence of 5′-AACAGUGUUCUUGCUCUAUAA-3′ (SEQ ID NO:1) by no more than 3, such as 3, 2, 1, or 0 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides, such as 15, 16, 17, 18, 19, 20, or 21, that differ from the nucleotide sequence of 5′-UUAUAGAGCAAGAACACUGUUUU-3′ (SEQ ID NO:2) by no more than 3, such as 3, 2, 1, or 0 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides, such as 3, 2, 1, or 0, that differ from the nucleotide sequence of 5′-UUAUAGAGCAAGAACACUGUUUU-3′ (SEQ ID NO:2) by no more than 3, such as 3, 2, 1, or 0 nucleotides. Five, for example, 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides, wherein the antisense strand comprises at least one modified nucleotide of formula (I) or formula (II) at the nucleotide positions 2–8 (e.g., 5–8) of the 5' region (counting from the 5' end); optionally, all nucleotides of the sense strand and other nucleotides of the antisense strand comprise nucleotide modifications selected from: 2′-O-methyl modification and 2′-fluorine modification and deoxynucleotide; wherein the sense strand comprises at least two 2′-fluorine modifications; wherein the antisense strand comprises at least two 2′-fluorine modifications; wherein the sense strand comprises four thiophosphate bonds; wherein the antisense strand comprises four thiophosphate bonds; optionally, wherein the sense strand is conjugated with a ligand. The double-stranded RNA molecule according to any one of claims 20-23, wherein the double-stranded RNA molecule is a dsRNA molecule that inhibits the expression of the hydroxy acid oxidase 1 (HAO1) gene, wherein the sense strand comprises at least 15 consecutive nucleotides, such as 15, 16, 17, 18, 19, 20, or 21, that differ from the nucleotide sequence of 5′-GAAUGUGAAAGUCAUCGACAA-3′ (SEQ ID NO:3) by no more than 3, such as 3, 2, 1, or 0 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides, such as 15, 16, 17, 18, 19, 20, or 21, that differ from the nucleotide sequence of 5′-UUGUCGAUGACUUUCACAUUCUG-3′ (SEQ ID NO:4 ... Five, for example, 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides, wherein the antisense strand comprises at least one modified nucleotide of formula (I) or formula (II) at the nucleotide positions 2–8 (e.g., 5–8) of the 5' region (counting from the 5' end); optionally, all nucleotides of the sense strand and other nucleotides of the antisense strand comprise nucleotide modifications selected from: 2′-O-methyl modification and 2′-fluorine modification and deoxynucleotide; wherein the sense strand comprises at least two 2′-fluorine modifications; wherein the antisense strand comprises at least two 2′-fluorine modifications; wherein the sense strand comprises four thiophosphate bonds; wherein the antisense strand comprises four thiophosphate bonds; optionally, wherein the sense strand is conjugated with a ligand. The double-stranded RNA molecule according to claim 24 or 25, wherein the double-stranded RNA molecule comprises paired sense strands and antisense strands as shown in Table 2B or Table 5. A pharmaceutical composition comprising a double-stranded RNA molecule according to any one of claims 20-26 and a pharmaceutically acceptable excipient. A gene silencing kit comprising a double-stranded RNA molecule according to any one of claims 20-26. The use of the compound according to any one of claims 1 to 19 is for preparing a double-stranded RNA molecule to silence a target gene and to suppress the off-target effects of the double-stranded RNA molecule. The use of the double-stranded RNA molecule according to any one of claims 20 to 26 for the preparation of a medicament for treating a disease.